Microcells

Investigations on  Bonamia ostreae and other related parasites 

The genus Bonamia includes small-size (2-3 μm) uninucleated protozoan parasites also called microcells. Two microcells are presently listed in Annex IV, Part II of the Directive 2006/88/EC: Bonamia ostreae which affects populations of European flat oyster Ostrea edulis and B. exitiosa which infects O. chilensis from New Zealand and O. angasi from Australia. Other known species of the genus are B. roughleyi infecting Saccostrea commercialis from Australia and B. perspora described in Ostreola equestris from North Carolina, USA. Besides these four species, no fully characterized Bonamia spp. were reported in O. chilensis in Chile, in O. puelchana in Argentina, in Crassostrea ariakensis in North Carolina, U.S.A. and in O. stentina in Tunisia.

 

 

The notification of Bonamia exitiosa in Spain in October 2007 and its detection in Adriatic Sea in Italy in Ostrea edulis outside mortality occurrence supports the apparent wide distribution of the parasite all around the world and questioned its impact on European flat oyster populations. The presence of both parasites B. exitiosa and B. ostreae in same areas and even in same individuals reinforces the need of specific diagnostic tools.

In 2014 the EURL has focused its activities on (1) the characterization of parasites of the genus Bonamia (2) the characterization of new genes of interest for taxonomic and diagnostic purposes and (3) some studies to better understand host-pathogen interactions.

1 - Characterisation of Bonamia parasites in Europe and Mediterranean basin

In October 2007, Spain notified the presence of Bonamia exitiosa in some flat oysters collected from Ria de Arousa, in Galicia. Subsequently, the EURL in collaboration with Italian colleagues detected Bonamia exitiosa in flat oysters Ostrea edulis from Manfredonia Gulf (Adriatic sea, Italy) (Narcisi et al. 2010).

In 2008, a workplan to characterise Bonamia parasites in Europe had been proposed by the EURL to NRLs with the support of the European Commission. The outcome of this study was to determine the spread of B. exitiosa in the EU.

As conclusions, so far Bonamia exitiosa has been detected and confirmed in 4 Member States: France, Italy, Spain and UK. However, where Bonamia exitiosa was not detected, it is not possible to conclude that these locations are free of Bonamia exitiosa. Mixed-infections (infections with both B. ostreae and B. exitiosa) in same location and even in same oyster were observed.

These last years, the EURL has investigated potential polymorphism among the parasites B. exitiosa detected in Europe and phylogenetic relationships between the European B. exitiosa and other related parasites detected worldwide. More particularly, a fragment of the gene encoding the actin was amplified and sequenced from samples collected in different countries in Europe. Obtained results confirmed that parasites identified as B. exitiosa in Europe and Mediterranean basin group together with B. exitiosa from New Zealand. These results question the origin of B. exitiosa in flat oyster populations in Europe.

 

2 - Characterization of new genes of interest for taxonomic and diagnostic purposes

Bonamia parasites are small no cultivable protozoans. Studies aiming at improving knowledge of their genomes are thus hampered by the difficulty to obtain sufficient amount of pure parasites. Until recently, the only characterized parts of the genome of Bonamia ostreae were some ribosomal RNA genes and two actin genes.

New assays were developed in order to improve these data and allowed identifying a gene encoding the cytosolic Heat Shock Protein 90 of Bonamia ostreae (Prado-Alvarez et al. 2013).

Additionally, some investigations allowed identifying part of the actin gene of B. exitiosa. This result allowed us including an additional target gene in our study on B. exitiosa infecting Ostrea edulis in Europe.

The EURL was involved in the Interreg IVB SUDOE project “Aquagenet” (development of biotechnological tools to develop and improve sustinable aquaculture in SUDOE area) and in this context has carried out RNA seq on RNA extracted from purified Bonamia ostreae cells. More than 300.10⁶ sequences were obtained, corresponding to 38.1 Gb. Less than 4% of contigs show alignement with oyster genome and transcriptome (blast -e 1e-5). Some sequences show best alignement with sequences from other protozoans including the soil-living amoeba Dictyostelium discoideum or the Apicomplexa parasite Plasmodium falciparum.

It is the first RNA seq approach carried out on a species of the genus Bonamia. Such study is more and more widely used to obtain sequences from unicellular and non cultivable parasites and has been recently applied on Mikrocytos mackini (Burki et al. 2013).

Some of these results were presented during international and national conferences:

Arzul Isabelle, Chollet Bruno, Klopp Christophe (2014). RNA seq for the identification of new sequences from Bonamia ostreae, a protozoan infecting flat oysters. National Shellfisheries Association 106th Annual Meeting, March 29 - April 2 2014, Jacksonville, United States.

Lapègue Sylvie, Arzul Isabelle, Boudry Pierre, Corporeau Charlotte, Gagnaire Pierre-Alexandre, Haffray Pierrick, Huvet Arnaud, Renault Tristan (2014). Les "omiques" pour mieux comprendre et améliorer la robustesse des huîtres face aux mortalités de juvéniles et d'adultes. Xe Colloque AGENAE "Génétique et génomique animale", 12 et 13 Mai 2014, Nantes.

3- Studies on interactions between flat oysters and Bonamia ostreae

The means to fight against bonamiosis are relatively limited. They are mainly based on oyster health surveillance to limit the spread of the disease. However, the use of predictive models of disease progression in infected area would help to improve stock management and minimize the impact of pathogens. Moreover, the development of resistant animals could help to revive this production. These different approaches require appropriate diagnostic tools, a good knowledge of the life cycle of the pathogen, and of interactions between the parasite and its host. In this context, studies are carried out to understand the interactions between the flat oyster Ostrea edulis and the parasite Bonamia ostreae, and particularly the molecular basis of the resistance to the parasite. Previous works suggested that modulation of apopotosis and decrease of phagocytosis could be involved in mechanisms related to resistance to bonamiosis. The role of apoptosis in the response of the bivalves against stress including pathogens like Bonamia ostreae is presently investigated in the laboratory and should allow identifying new targets for control measures.

Apoptosis is considered as a defense mechanism against stress factors including pathogens and studies carried out on molluscs have shown that apoptosis takes an important place in protection against protozoan parasites. In the context of experimental contacts between hemocytes from flat oysters and Bonamia ostreae isolated from infected oysters, the different tested cellular activities appeared increased including phosphatidyl serine externalisation, intracytoplasmic calcium concentration, mitochondrial membrane potential, caspase activities and DNA fragmentation. Our results show that apoptosis is an important mechanism developed by the flat oyster against bonamiosis.

 

Some of these results were published and presented at international conferences:

Engelsma Marc, Culloty Sarah, Lynch Sharon, Arzul Isabelle, Carnegie Ryan (2014). Bonamia parasites: a rapidly changing perspective on a genus of important mollusc pathogens. Diseases of Aquatic Organisms, 110(1-2), 5-23.

Gervais Ophelie, Chollet Bruno, Renault Tristan, Arzul Isabelle (2014). Modulation of apoptosis by stress factors including the protozoan parasite Bonamia ostreae in the flat oyster Ostrea edulis. National Shellfisheries Association 106th Annual Meeting, March 29 - April 2 2014, Jacksonville, United States.

Other information

You can find more information on these pathogens on:

1) OIE website, France (OIE Manual of Diagnostic Tests for Aquatic animals, 2012):

Table of contents : http://www.oie.int/eng/normes/fmanual/A_summry.htm

2) DFO website, Canada (Synopsis of Infectious Diseases and Parasites of Commercially Exploited Shellfish):

Table of contents:  http://www.pac.dfo-mpo.gc.ca/sci/shelldis/title_e.htm