Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 3.1.1.

BONAMIOSIS
(Bonamia exitiosus, B. ostreae
and Mikrocytos roughleyi)

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GENERAL INFORMATION

Bonamiosis here refers only to the diseases in oysters caused by Bonamia ostreae in the Northern Hemisphere, and by Bonamia exitiosus and Mikrocytos roughleyi in the Southern Hemisphere. If detected outside the known range of Mikrocytos roughleyi and Bonamia spp., electron microscopy or DNA-based assays, when available, must be used to identify and distinguish the detected organism from other microcell species (Bonamia ostreae, Bonamia exitiosus, Mikrocytos mackini and M. roughleyi). The presence of these pathogens in any bivalve should be regarded as potentially serious and the OIE Reference Laboratory should be consulted.
 
Bonamiosis is caused by the haplosporidians Bonamia ostreae, B. exitiosus and Mikrocytos roughleyi (3-5, 8, 11, 17, 21). Bonamiosis is also known as microcell disease, haemocyte disease of flat oysters (B. ostreae), haemocyte disease of dredge oysters (Bonamia exitiosus) or winter mortality (Mikrocytos roughleyi).
 
Bonamia ostreae occurs naturally in Ostrea edulis and O. conchaphila (= O. lurida), and can infect O. puelchana, O. angasi and Ostrea chilensis (= Tiostrea chilensis, T. lutaria) when moved into endemic zones (12, 13, 20). Bonamia exitiosus occurs in O. angasi, O. denselammellosa and T. chilensis (8). Species of Ostrea and Tiostrea and other ostreids (Crassostrea rivularis = C. arakensis) (6) should be considered to be potentially susceptible to Bonamia spp. Mikrocytos roughleyi infects the Sydney rock oyster Saccostrea glomerata (commercialis).
 
The geographical distribution of B. exitiosus is: New Zealand (around South Island and lower North Island) (9) and Australia (Western Australia, Victoria and Tasmania). Bonamia ostreae has been reported from France, Ireland, Italy, the Netherlands, Spain, the United Kingdom (excluding Scotland), and the United States of America (California, Maine and Washington State) (2, 10, 18, 19, 22, 23), but is now thought to be absent from Denmark. Mikrocytos roughleyi occurs in New South Wales, Australia.
 
Bonamiosis is a lethal infection of the haemocytes of flat oysters, sometimes accompanied by yellow discoloration and extensive lesions on the gills and mantle (21). However, most of the infected oysters appear normal. Lesions occur in the connective tissue of the gills, mantle, and digestive gland. These intrahaemocytic protistans quickly become systemic with overwhelming numbers of parasites coinciding with the death of the oysters. In highly susceptible hosts, bonamiosis is a lethal infection of the haemocytes. Some evidence suggests that the pathology caused by Bonamia spp. depends on which host species and population is infected. For example, B. exitiosus in oysters from Tasmania were highly epitheliotropic and associated with focal abscesses in some populations without evidence of systemic spread. Mikrocytos roughleyi is associated with focal abcess-type lesions in the gill, connective and gonadal tissues and alimentary tract (11). It induces a systemic intracellular infection in the haemocytes (but never in the connective tissue cells).
 
Bonamia spp. may occur throughout the year, but prevalence and intensity of infection tend to increase during the warm season (7, 12, 15). There is a seasonal variation in infection by B. ostreae with the highest prevalence occurring in September in the Northern Hemisphere. In the Southern Hemisphere, B. exitiosus shows the highest prevalence from January to April with the parasite being barely detectable in September and October. The prepatent period for B. ostreae and B. exitiosus is usually 3-4 months but can be up to 5 months. In the Southern Hemisphere, the disease caused by M. roughleyi is associated with low temperatures and high salinities. It can kill up to 70% of mature Sydney rock oysters in their third winter before marketing, and has a prepatent period of 2.5 months (11).
 
Bonamiosis can be transmitted experimentally by cohabitation or inoculation (14).
 
Examination of stained tissue sections and tissue imprints of susceptible organs are the recommended methods for screening (1, 24). For diagnosis, the recommended guidelines for sampling are those stated in Chapter 1.1.4 and Chapter I.2. of this Aquatic Manual. However, the disease may be not detectable by the usual methods during the first 5 months of infection.
 

EXAMINATION PROCEDURES

1.	Screening Methods
	1.1.	Histology
		General histological procedures are detailed in Chapter I.2. of this Aquatic Manual. A transversal 'steak' is cut dorso-ventrally through the soft tissues and then immediately placed in fixative medium, or (depending on the size of the animal) the whole animal, removed from the shell, can be fixed with a fixative, such as Davidson's solution, to provide optimal parasite definition.
		Several nonspecific stains, e.g. haematoxylin-eosin (H& E), enable Bonamia and Mikrocytos to be visualised. It is recommended that a section through the cardiac cavity, digestive gland and gills be examined per oyster.
		Bonamia spp. (2-5 µm in size) occurs within the haemocytes or freely in connective tissue or sinuses of the gill, gut or mantle epithelium. However, the parasite has to be observed inside the haemocytes for a positive diagnosis to be made to avoid false-positive results.
		In abscesses, Mikrocytos roughleyi may be observed within the haemocytes as 1-2 µm organisms with spherical nucleus greater than 1 µm containing bipolar or eccentric nucleolar structures. When present, a cytoplasmic vacuole may displace the nucleus to the periphery of the cell.
		Microcytos roughleyi differs from M. mackini in that all stages of M. roughleyi are present in the haemocytes, and M. mackini occurs usually in the vesicular connective tissue cells on the periphery of the lesions or in myocytes of the adductor muscle and occasionally within haemocytes.
	1.2.	Cytological examination: tissue imprints
		Make impression smears of oyster spat or heart tissue (preferably ventricle) on a histological slide. The slides are air-dried and fixed in methanol. The prepared imprints are stained using a commercially available blood-staining kit, in accordance with the manufacturer's instructions. After staining, the slides are rinsed in tap water, allowed to dry completely in cold or warm air, and mounted with a cover-slip using an appropriate synthetic resin.
		The parasite (2-5 µm in size) has basophilic cytoplasm and an eosinophilic nucleus (colours may vary with stain used). It may be observed inside or outside the haemocytes. An observation time of 10 minutes per slide is sufficient (under oil immersion, x800-1000 magnification). The organisms are enlarged by this method compared with those observed in histology. Heart smears are inappropriate in stocks where the infection, if present, remains largely epitheliotropic.
2.	Presumptive Diagnostic Methods
	Cytological examination and histopathology, as described above, may be used.
3.	Confirmatory Identification of the Pathogen
	3.1.	Transmission electron microscopy examination
		Transmission electron microscopy procedures are described in Chapter I.2. of this Aquatic Manual.
		Differences exist between Bonamia exitiosus forms and B. ostreae: there are fewer haplosporosomes, mitochondrial profiles and lipoid bodies in dense forms of B. ostreae, smaller nucleus and cytoplasm ratio (8, 17, 21).
		Plasmodial forms of Bonamia exitiosus are distinguished by their size (4.0-4.5 µm), irregular cellular and nuclear outline, amorphous cytoplasmic inclusions (multi-vesicular bodies), and arrays of Golgi-like smooth endoplasmic reticulum. Forms of intermediate cytoplasmic density are more electron dense than the plasmodial forms and slightly smaller in diameter (3.0-3.5 µm). Haplosporosomes are formed from Golgi/nuclear material complexes and are similar in construction and structure to some viruses.
		Ultrastructural morphology differentiates M. mackini from Bonamia spp.; the nucleolus of M. mackini is located towards the centre of the nucleus while that of B. ostreae has an eccentric location and there is an apparent lack of mitochondria in M. mackini.

REFERENCES

1.	Bachere E., Durand J.-L. & Tige G. (1982). Bonamia ostreae (Pichot et al., 1979) parasite de l'huitre plate : comparaison de deux méthodes de diagnostic. Cons. Int. Exploit. Mer, 28, 1-10.
2.	Bucke D., Hepper B., Key D. & Bannister C.A. (1984). A report on Bonamia ostreae in Ostrea edulis in the UK. Cons. Inter. Explor. Mer, CM 1984/K, 9, 1-7.
3.	Carnegie R., Barber B.J., Culloty S.C., Figueras A.J. & Distel D.L. (2000). Development of a PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the Haplosporidia. Dis. Aquat. Org., 42, 199-206.
4.	Cochennec N., Le Roux F., Berthe F. & Gérard A. (2000). Detection of Bonamia ostreae based on small subunit ribosomal probe. J. Invertebr. Pathol., 76, 26-32.
5.	Cochennec N., Reece K.S., Berthe F.C.J. & Hine P.M. (2003). Revisiting Mikrocytos roughleyi taxonomic affiliation points to the genus Bonamia (Haplosporidia). Dis. Aquat. Org., (in press).
6.	Cochennec N., Renault T., Boudry P., Chollet B. & Gerard A. (1998). Bonamia-like parasite found in the Suminoe oyster Crassostrea rivularis reared in France. Dis. Aquat. Org., 34, 193-197.
7.	Culloty S.C. & Mulcahy M.F. (1996). Season-, age-, and sex-related variation in the prevalence of bonamiosis in flat oysters (Ostrea edulis L.) on the south coast of Ireland. Aquaculture, 144, 53-63.
8.	Dinamani P., Hine P.M. & Jones J.B. (1987). Occurrence and characteristics of the haemocyte parasite Bonamia sp. in the New Zealand dredge oyster Tiostrea lutaria. Dis. Aquat. Org., 3, 37-44.
9.	Doonan I.J., Cranfield H.J. & Michael K.P. (1994). Catastrophic reduction of the oyster, Tiostrea chilensis (Bivalvia: Ostreidae), in Foveaux strait, New Zealand, due to infestation by the protistan Bonamia sp. NZ J. Mar. Freshwater Res., 28, 335-344.
10.	Elston R.A., Farley C.A. & Kent M.L. (1986). Occurrence and significance of bonamiasis in European flat oysters Ostrea edulis in North America. Dis. Aquat. Org., 2, 49-54.
11.	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.
12.	Grizel H. (1985). Etudes des récentes épizooties de l'huitre plate Ostrea edulis L. et de leur impact sur l'ostreiculture bretonne. Thèse de doctorat, Université des Sciences et Techniques de Languedoc, Montpellier, France.
13.	Grizel H., Comps M., Raguennes D., Leborgne Y., Tige G. & Martin A.G. (1983). Bilan des essais d'acclimation d'Ostrea chilensis sur les côtes de Bretagne. Rev. Trav. Inst. Peches Marit., 46, 209-225.
14.	Hervio D., Bachere E., Boulo V., Cochennec N., Vuillemin V., Le Coguic Y., Cailletaux G., Mazurie J. & Mialhe E. (1985). Establishment of an experimental infection protocol for the flat oyster Ostrea edulis with the intrahaemocytic protozoan parasite Bonamia ostreae: application in the selection of parasite-resistant oyster. Aquaculture, 132, 183-194.
15.	Hine P.M. (1991). The annual pattern of infection by Bonamia sp. in New Zealand flat oysters, Tiostrea chilensis. Dis. Aquat. Org., 11, 163-171.
16.	Hine P.M., Bower S.M., Meyer G.R., Cochennec-Laureau N. & Berthe F. (2001). Ultrastructure of Mykrocytos mackini, the cause of Denman Island disease in oysters Crassostrea spp. and Ostrea spp. in British Columbia, Canada. Dis. Aquat. Org., 45, 215-227.
17.	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.
18.	McArdle J.F., McKiernan F., Foley H. & Jones D.H. (1991). The current status of Bonamia disease in Ireland. Aquaculture, 93, 273-278.
19.	Montes J. & Melendez I. (1987). Données sur la parasitose de Bonamia ostreae chez l'huitre plate de Galice, côte Nord-Ouest de l'Espagne. Aquaculture, 67, 195-198.
20.	Pascual M., Martin A.G., Zampatti E., Coatanea D., Defossez J. & Robert R. (1991). Testing of the Argentina oyster, Ostrea puelchana, in several French oyster farming sites. Cons. Inter. Explor. Mer, CM 1991/K30.
21.	Pichot Y., Comps M., Tige G., Grizel H. & Rabouin M.A. (1979). Recherches sur Bonamia ostreae gen. n., sp. n., parasite nouveau de l'huitre plate Ostrea edulis L. Rev. Trav. Inst. Peches Marit., 43, 131-140.
22.	Tige G., Grizel H., Cochennec N. & Rabouin M.A. (1984). Evolution de la situation épizootiologique en Bretagne en 1983 suite au développement de Bonamia ostreae. Cons. Inter. Explor. Mer, CM 1984/F, 14, 1-10.
23.	Van Banning P. (1987). Further results of the Bonamia ostreae challenge tests in Dutch oyster culture. Aquaculture, 67, 191-194.
24.	Zabaleta A. & Barber B.J. (1996). Prevalence, intensity and detection of Bonamia ostreae in Ostrea edulis L in the Damariscotta River area, Maine. J. Shellfish Res., 15, 395-400.

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