Living with bonamiasis: Irish research since 1987

The main oyster species produced in Ireland up to the 1980's was the European flat oyster Ostrea edulis. However, since then, production of this species has been severely affected by the presence of the protistan Bonamia ostreae, which was diagnosed in a population of flat oysters on the south coast following heavy mortalities. Research has been ongoing since the first diagnosis in Ireland and has concentrated on aspects of the biology of both the host and the parasite. In recent years research has concentrated on screening populations of oysters to identify any with reduced susceptibility to the parasite.     

Bonamia exitiosa (Haplosporidia) observed infecting the European flat oyster Ostrea edulis cultured on the Spanish Mediterranean coast

Bonamia exitiosa and Bonamia ostreae are parasites that reproduce within the haemocytes of several oyster species. In Europe, the host species is the flat oyster Ostrea edulis. The parasite B. ostreae has been responsible for mortalities since the late 1970s throughout the European Atlantic coast. B. exitiosa was first detected, in 2007, on this continent in flat oysters cultured in Galicia (NW Spain). Since then, the parasite has also been detected in France, Italy and the United Kingdom. The bays of the Ebro Delta in the south of Catalonia represent the main bivalve culture area in the Mediterranean coast of Spain. Previous information from the area includes reports of several flat oyster pathogens, including the notifiable parasite Marteilia refringens. However, the status with regard to Bonamia parasites was uncertain. In the present study, a Bonamia parasite was observed in flat oysters cultured in the Alfacs Bay of the Ebro Delta by histology and real-time PCR. PCR-RFLP and sequencing suggested the presence of B. exitiosa. Finally, phylogenetic analyses of the studied Bonamia isolates corroborated B. exitiosa infection. M. refringens was also observed in the same oyster batch, and co-infection with both parasites was also detected. This is the first detection of B. exitiosa, in Catalonia and the Spanish Mediterranean coast. The impact of the parasite on the Mediterranean flat oyster activity needs to be urgently addressed.     

Severe apicomplexan infection in the oyster Ostrea chilensis: a possible predisposing factor in bonamiosis

Histological examination of 6455 oysters Ostrea chilensis from Foveaux Strait south of New Zealand over a 5 yr period showed >85% contained apicomplexan zoites, irrespective of season. Zoites occurred around the haemolymph sinuses and the digestive diverticulae at all intensities of infection; occurrence in the sub-epithelium, Leydig tissue and gills/mantle increased with increasing intensity of infection. Many (>35%) oysters were heavily infected, and most of them had severely damaged tissues. Heavy infections affected gametogenesis; 1% of lightly infected oysters had empty gonad follicles lacking germinal epithelium compared with 2% of moderately infected oysters and 9% of heavily infected oysters. Of oysters with empty gonad follicles, 75% were heavily infected with zoites. The parasite spread from the haemolymph sinuses and moved between Leydig cells, causing their dissociation and lysis. Some zoites were intracellular in Leydig cells. Lesions contained many haemocytes phagocytosing zoites, leading to haemocyte lysis and causing a haemocytosis. Fibrosis occurred to repair lesions in a few oysters. The zoites had a typical apical complex with 2 polar rings and 84 sub-pellicular microtubules. Prevalence and intensity of concurrent Bonamia exitiosus infection was related to the intensity of zoite infection, with only 3.8% of B. exitiosus infections occurring in the absence of zoites, 20.0% occurring in light zoite infections, 30.9% in moderate zoite infections, and 45.4% when oysters were heavily infected with zoites. The converse was not the case, as 75.3% of zoite infections occurred in the absence of B. exitiosus infection, including 51.1% of moderate to heavy zoite infections. There was a statistically significant association between intensities of B. exitiosus and of zoites (p < 0.0001). Zoites may increase the susceptibility of oysters to B. exitiosus by occupying and destroying haemocytes, and by destroying connective tissue cells and utilising host glycogen reserves. The parasite may be heteroxenous, with other stages in the terebellid polychaete Pseudopista rostrata.     

Investigating the possible role of benthic macroinvertebrates and zooplankton in the life cycle of the haplosporidian Bonamia ostreae

Bonamia ostreae is a protistan parasite of the European flat oyster, Ostrea edulis. Though direct transmission of the parasite can occur between oysters, it is unclear if this represents the complete life cycle of the parasite, and the role of a secondary or intermediate host or carrier species cannot be ruled out. In this preliminary study, benthic macroinvertebrates and zooplankton from a B. ostreae-endemic area were screened for the presence of parasite DNA, using polymerase chain reaction (PCR). Eight benthic macroinvertebrates and nineteen grouped zooplankton samples gave positive results. Certain species, found positive for the parasite DNA, were then used in laboratory transmission trials, to investigate if they could infect na?ve oysters. Transmission of B. ostreae was effected to two na?ve oysters cohabiting with the brittle star, Ophiothrix fragilis.     

Systematic evaluation of factors controlling Perkinsus marinus transmission dynamics in lower Chesapeake Bay

The transmission of Perkinsus marinus in eastern oysters Crassostrea virginica in relation to water temperature, host oyster mortality, and water-column abundance of anti-P. marinus antibody-labeled cells was systematically examined for 20 mo at a site in the lower York River, Virginia, USA. Uninfected sentinel oysters were naturally exposed to the parasite at 2 wk intervals throughout the course of the study to determine the periodicity and rates of parasite transmission. The timing and magnitude of disease-associated oyster mortalities in a local P. marinus-infected oyster population were estimated by monitoring a captive subset of the local oyster population. Flow cytometric immunodetection methods were employed to estimate the abundance of P. marinus cells in water samples collected 3 times each week. The acquisition of P. marinus infections by na?ve sentinel oysters occurred sporadically at all times of the year; however, the highest incidence of infection occurred during the months of August and September. This window of maximum parasite transmission coincided with the death of infected hosts within the captive local oyster population. Counts of antibody-labeled cells ranged from 10 to 11900 cells l(-1), with the highest abundances in July and August coincident with maximum summer temperatures. A statistically significant relationship between water-column parasite abundance and infection-acquisition rate was not observed; however, highest parasite-transmission rates in both years occurred during periods of elevated water-column abundance of parasite cells. These results support the prevailing model of P. marinus transmission dynamics by which maximum transmission rates are observed during periods of maximum P. marinus-associated host mortality. However, our results also indicate that transmission can occur when host mortality is low or absent, so alternative mortality-independent dissemination mechanisms are likely. The results also suggest that atypically early-summer oyster mortality from Haplosporidium nelsoni infection, at a time when infections of P. marinus are light, has a significant indirect influence on P. marinus transmission dynamics. Elimination of these hosts prior to late-summer P. marinus infection-intensification effectively reduces the overall number of P. marinus cells disseminated.     

Detection of the oyster parasite Bonamia ostreae by fluorescent in situ hybridization

Bonamia ostreae is an economically significant protistan parasite of the flat oyster Ostrea edulis in Europe and North America. Management of this parasite depends partly upon its reliable identification in wild and aquacultured oyster populations, but B. ostreae is small and difficult to detect by traditional microscopic methods. We designed a fluorescent in situ hybridization (FISH) assay to sensitively detect B. ostreae in standard histopathological sections of B. ostreae-infected oysters using fluorescently labeled DNA oligonucleotide probes. Hybridization using a cocktail of 3 presumptively B. ostreae-specific, fluorescein iso(thio)cyanate (FITC)-labeled oligonucleotides produced an unambiguous staining pattern of small green rings inside infected oyster hemocytes that was easily distinguished from host tissue background. This pattern is diagnostic for B. ostreae. A negative control cocktail of oligonucleotides containing 2 mismatches relative to target sequences, on the other hand, failed to hybridize at all. B. ostreae-specific probes did not cross-react with a related protist, Haplosporidium nelsoni.     

Strong seasonality of Bonamia sp. infection and induced Crassostrea ariakensis mortality in Bogue and Masonboro Sounds, North Carolina, USA

Asian oyster Crassostrea ariakensis is being considered for introduction to Atlantic coastal waters of the USA. Successful aquaculture of this species will depend partly on mitigating impacts by Bonamia sp., a parasite that has caused high C. ariakensis mortality south of Virginia. To better understand the biology of this parasite and identify strategies for management, we evaluated its seasonal pattern of infection in C. ariakensis at two North Carolina, USA, locations in 2005. Small (<50 mm) triploid C. ariakensis were deployed to upwellers on Bogue Sound in late spring (May), summer (July), early fall (September), late fall (November), and early winter (December) 2005; and two field sites on Masonboro Sound in September 2005. Oyster growth and mortality were evaluated biweekly at Bogue Sound, and weekly at Masonboro, with Bonamia sp. prevalence evaluated using parasite-specific PCR. We used histology to confirm infections in PCR-positive oysters. Bonamia sp. appeared in the late spring Bogue Sound deployment when temperatures approached 25 degrees C, six weeks post-deployment. Summer- and early fall-deployed oysters displayed Bonamia sp. infections after 3-4 weeks. Bonamia sp. prevalences were 75% in Bogue Sound, and 60% in Masonboro. While oyster mortality reached 100% in late spring and summer deployments, early fall deployments showed reduced (17-82%) mortality. Late fall and early winter deployments, made at temperatures <20 degrees C, developed no Bonamia sp. infections at all. Seasonal Bonamia sp. cycling, therefore, is influenced greatly by temperature. Avoiding peak seasonal Bonamia sp. activity will be essential for culturing C. ariakensis in Bonamia sp.-enzootic waters.     

Detection of Bonamia ostreae based on small subunit ribosomal probe

Bonamia ostreae is a protozoan parasite of the flat oyster, Ostrea edulis, which has caused significant loss of oysters in Europe over the last decade. B. ostreae was purified from infected flat oysters and DNA was extracted. The nearly complete small subunit rDNA gene of B. ostreae was amplified using universal oligonucleotides and the PCR product was cloned and sequenced. BLAST research with this sequence revealed similarities to Haplosporidium nelsoni, Haplosporidium costale, and Minchinia teredinis. These data suggest that B. ostreae may be included in the genus Haplosporidium. Specific B. ostreae primers were designed for labeling, by PCR, a probe. This probe was successfully used by in situ hybridization to detect B. ostreae in infected fiat oysters, thus confirming the accuracy of this SSU rDNA sequence. The probe lead also to the detection of Bonamia sp. in infected Tiostrea chilensis and H. nelsoni in infected Crassostrea virginica but not Mikrocytos mackini infected Crassostrea gigas. These primers were also used to detect B. ostreae from infected oyster tissues by PCR. This B. ostreae SSU rDNA gene sequence provides genetic information as a first step toward elucidation of the taxonomic boundaries among the microcell organisms. Moreover, the development of DNA detection assays will be valuable specific diagnostic tools.     

Pathology of bonamiasis

Bonamiasis is a serious disease affecting the oyster industry, particularly in Europe where it has caused catastrophic losses since its appearance in 1979. The causative agent is now recognized as a protistan, Bonamia ostreae(1,2), which aggressively infects Ostrea species to cause clinical disease that rapidly spreads through oyster stocks, usually causing heavy mortalities. The economic importance o f this parasite has generated wide interest but, as David Bucke discusses here, neither its taxonomic status nor its life cycle are clearly understood and there is much to be learn about its pathogenesis and possible means of control.     

Immune parameters of QX-resistant and wild caught Saccostrea glomerata hemocytes in relation to Marteilia sydneyi infection

Sydney rock oysters (SRO) Saccostrea glomerata suffer mass mortalities during summer and autumn as a result of infection by a protozoan parasite Marteilia sydneyi (QX disease). Mass selected disease resistant (QXR) lines have been used with some success in affected estuaries in recent years, with resistance attributed to oxidative defense systems. However, the role of hemocytes in resistance to QX by SRO has not been fully explored. In the present study, fifty QXR and fifty wild caught (WC) oysters were collected from a lease at Pimpama River during a QX outbreak in January 2011. Hemocytes characteristics (type, morphology) and functions (mortality, phagocytosis and oxidative activity) from both oyster lines were analyzed by flow cytometry in the context of infection intensity and parasite viability (determined histologically). Amongst the QXR oysters, 20% were diseased containing viable parasite, 74% had killed M. sydneyi and 6% were uninfected. In contrast, 86% of WC oysters were diseased, 2% had killed M. sydneyi and 12% were healthy. Significant differences in hemocyte number and physiology between the two oyster lines were found (ANOVA). Phagocytosis rate and the mean oxidative activity per cell were similar between both oyster lines. Higher numbers of infiltrating and circulating hemocytes, higher percentage of circulating granulocytes, their higher size and complexity in QXR oysters, and the production of reactive oxygen species were associated with the ability to kill the parasite. High abundance of M. sydneyi in the digestive tubule epithelium of both oyster lines implied inability to kill the parasite at the beginning of the infection. However, QXR oysters had the ability to kill M. sydneyi at the stage of sporangiosorae in the epithelium of digestive tubules. The similar phagocytic ability of hemocytes from both oyster lines, the size of the parasite at this infection stage, and its localization suggested that encapsulation is likely to be the main process involved in the eradication of M. sydneyi by QXR oysters.     

Spore ornamentation of Minchinia occulta n. sp. (Haplosporidia) in rock oysters Saccostrea cuccullata (Born, 1778)

A Minchinia sp. (Haplosporidia: Haplosporidiidae) parasite was identified infecting rock oysters and morphologically described by Hine and Thorne (2002) using light microscopy and transmission electron microscopy (TEM). The parasite was associated with up to 80% mortality in the host species and it is suspected that the parasite would be a major impediment to the development of a tropical rock oyster aquaculture industry in northern Western Australia. However, attempts to identify the parasite following the development of a specific probe for Haplosporidium nelsoni were unsuccessful. The SSU region of the parasite's rRNA gene was later characterized in our laboratory and an in situ hybridization assay for the parasite was developed. This study names the parasite as Minchinia occulta n sp. and morphologically describes the parasite using histology, scanning electron microscopy and transmission electron microscopy. The non-spore stages were unusual in that they consisted primarily of uninucleate stages reminiscent of Bonamia spp. The parasite's spores were ovoid to circular shaped and measured 4.5 microm-5.0 microm x 3.5-4.1 microm in size. The nucleus of the sporoplasm measured 1.5-2.3 microm and was centrally located. The spores were covered in a branching network of microtubule-like structures that may degrade as the spore matures.     

Comparison of in vitro-cultured and wild-type Perkinsus marinus. II. Dosing methods and host response

Endoparasites must breach host barriers to establish infection and then must survive host internal defenses to cause disease. Such barriers may frustrate attempts to experimentally transmit parasites by 'natural' methods. In addition, the host's condition may affect a study's outcome. The experiments reported here examined the effect of dosing method and host metabolic condition on measures of virulence for the oyster parasite Perkinsus marinus. Oysters, Crassostrea virginica, were challenged with wild-type and cultured forms of P. marinus via feeding, shell-cavity injection, gut intubation and adductor-muscle injection. For both parasite types, adductor-muscle injections produced the heaviest infections followed by shell-cavity injection, gut intubation, and feeding. There was no difference in parasite burdens between oysters fed cultured cells by acute vs chronic dosing, and parasite loads stabilized over time, suggesting a dynamic equilibrium between invasion and elimination. P. marinus distribution among tissues of challenged oysters indicated that parasites invaded the mantle and gill, as well as the gut, which has been considered the primary portal of entry. Frequency distributions of P. marinus in oysters challenged with 3 different culture phases indicated an aggregated distribution among hosts and suggested that stationary-phase parasites were easiest for the oyster to control or eliminate and log-phase parasites were the most difficult. Host metabolic condition also affected experimental outcomes, as indicated by increased infection levels in oysters undergoing spawning and/or exposed to low oxygen stress.     

Supplementation of Perkinsus marinus cultures with host plasma or tissue homogenate enhances their infectivity

The protozoan oyster parasite Perkinsus marinus can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of P. marinus-susceptible (Crassostrea virginica) and -tolerant (Crassostrea gigas, Crassostrea ariakensis) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.     

Lysozyme activity and protein concentration in the haemolymph of the flat oyster Ostrea edulis (L.)

Lysozyme activity and protein concentration in the haemolymph of the flat oyster Ostrea edulis were investigated. These biochemical constituents of the haemolymph could be an indication of the physiological condition and vitality of the defence system of an animal. Haemolymph protein and lysozyme in oysters were examined over an 18 month period to determine their relationship with the strain of oyster, the season, the site, and parasitism by Bonamia ostreae. Haemolymph protein concentration exhibited seasonal fluctuations and varied between strains. Levels of protein in oysters highly infected with B. ostreae were slightly depressed but not significantly so. Haemolymph lysozyme varied greatly between individuals but no correlation was found between lysozyme levels and infection of oysters by B. ostreae.     

Detection of Minchinia sp., in rock oysters Saccostrea cuccullata (Born, 1778) using DNA probes

Haplosporidian parasites infect various invertebrate hosts including some commercially important shellfish. Haplosporidium nelsoni (along with Perkinsus marinus) has severely affected Eastern oyster production on the eastern seaboard of the United States and flat oyster production in Europe has been severely impacted by Bonamia ostreae. These parasites are also often present at a very low prevalence and there are a variety of morphologically similar species that can be difficult to differentiate during cytological or histological diagnosis hence the need to develop specific tests. Recently, a Minchinia sp. was described affecting rock oysters (Saccostrea cuccullata) in north Western Australia. In this study, two in situ hybridisation (ISH) assays and a PCR assay have been developed and optimised for use in investigating these parasites. The first ISH assay used a 166bp polynucleotide probe while the second used a 30bp oligonucleotide probe. The specificity of each ISH assay was assessed by applying each probe to a variety of haplosporidian (5), a paramyxian (1) or ciliophora (1) parasites. The polynucleotide probe produced strong hybridisation signals against all of the haplosporidian parasites tested (Minchinia sp., Minchinia teredinis, Bonamia roughleyi, H. nelsoni and Haplosporidium costale) while the oligonucleotide probe recognised only the Minchinia sp. Both probes failed to detect the paramyxian (Marteilia sp.) or the Rhynchodid-like ciliate. The PCR assay amplifies a 220bp region and detected Minchinia sp. DNA from 50ng of genomic DNA extracted from the tissues of infected oysters and 10fg of amplified Minchinia sp. DNA. The assay did not react to oysters infected with H. nelsoni or H. costale. The ability of the PCR and oligonucleotide ISH assay to diagnose Minchinia sp. infected oysters was compared to histological examination from a sample of 56 oysters. The PCR assay revealed 26 infections while histological examination detected 14 infections. The oligonucleotide ISH assay detected 29 infections. The oligonucleotide ISH and PCR assays were found to be significantly more sensitive than histology for detecting the parasite.     

Supplementation of Perkinsus marinus Cultures with Host Plasma or Tissue Homogenate Enhances Their Infectivity

The protozoan oyster parasite Perkinsus marinus can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of P. marinus-susceptible (Crassostrea virginica) and -tolerant (Crassostrea gigas, Crassostrea ariakensis) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.     

Molecular characterisation of a haplosporidian parasite infecting rock oysters Saccostrea cuccullata in north Western Australia

A haplosporidian parasite was identified in rock oysters (Saccostrea cuccullata Born, 1778) from the Montebello Islands (latitude -20.4'S longitude 115.53'E) off the northern coast of Western Australia by histopathological examination, PCR amplification and DNA sequencing of a segment of the SSU region of the parasite's rRNA gene. An oligonucleotide probe was constructed from the parasite's SSU rRNA gene in order to confirm its presence by in situ hybridisation. The parasite was disseminated throughout the gonad follicles of the host and to a lesser extent in the gills. The only parasite life stages thus far observed in this study were a uninucleate naked cell assumed to be a precursor to multinucleate plasmodial stages and a binucleate plasmodial stage. Whilst no parasite spores were detected in affected rock oysters, a phylogenetic analysis of the SSU region of the parasite's rRNA gene indicates the parasite belongs to the genus Minchinia. A PCR and in situ hybridisation assay for the Minchinia sp. was used to identify haplosporidians described by Hine and Thorne [Hine, P.M.., Thorne, T., 2002. Haplosporidium sp. (Haplosporidia: Haplosporidiidae) associated with mortalities among rock oysters Saccostrea cuccullata in north Western Australia. Dis. Aquat. Organ. 51, 123-13], in archived rock oyster tissues from the same coastline.     

Combining Two-Stage Testing and Interval Mapping Strategies to Detect QTL for Resistance to Bonamiosis in the European Flat Oyster Ostrea edulis

We have identified quantitative trait loci (QTL) in the flat oyster (Ostrea edulis) for resistance to Bonamia ostreae, a parasite responsible for the dramatic reduction in the aquaculture of this species. An F₂ family from a cross between a wild oyster and an individual from a family selected for resistance to bonamiosis was cultured with wild oysters injected with the parasite, leading to 20% cumulative mortality. Selective genotyping of 92 out of a total of 550 F₂ progeny (i.e., 46 heavily infected oysters that died and 46 parasite-free oysters that survived) was performed using 20 microsatellites and 34 amplification fragment length polymorphism primer pairs. Both a two-stage testing strategy and QTL interval mapping methods were used. The two-stage detection strategy had a high power with a low rate of false positives and identified nine and six probable markers linked to genes of resistance and susceptibility, respectively. Parent-specific genetic linkage maps were built for the family, spanning ten linkage groups (n = 10) with an observed genome coverage of 69–84%. Three QTL were identified by interval mapping in the first parental map and two in the second. Good concordance was observed between the results obtained after the two-stage testing strategy and QTL mapping.