Loop-Mediated Isothermal Amplification for the Fast Detection of Bonamia ostreae and Bonamia exitiosa in Flat Oysters.

The haplosporidian parasites Bonamia ostreae (BO) and B. exitiosa (BE) are serious oyster pathogens. Two independent laboratories evaluated fluorescence real-time loop-mediated isothermal amplification (LAMP) assays for rapidly detecting these parasites. Specific LAMP assays were designed on the BO actin-1 and BE actin genes. A further generic assay was conceived on a conserved region of the 18S gene to detect both Bonamia species. The optimal reaction temperature varied from 65 to 67 °C depending on the test and instrument. Melting temperatures were 89.8-90.2 °C, 87.0-87.6 °C, and 86.2-86.6 °C for each of the BO, BE, and generic assays. The analytical sensitivity of these assays was 50 copies/µL in a 30 min run. The BO and BE test sensitivity was ~1 log lower than a real-time PCR, while the generic test sensitivity was similar to the real-time PCR. Both the BO and BE assays were shown to be specific; however, the generic assay potentially cross-reacts with Haplosporidium costale. The performance of the LAMP assays evaluated on samples of known status detected positives within 7-20 min with a test accuracy of 100% for the BO and generic tests and a 95.8% accuracy for BE. The ease of use, rapidity and affordability of these tests allow for field deployment.

Species which may act as vectors or reservoirs of diseases covered by the Animal Health Law: Listed pathogens of molluscs.

Vector or reservoir species of five mollusc diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review, to support a possible updating of Regulation (EU) 2018/1882. Mollusc species on or in which Mikrocytos mackini, Perkinsus marinus, Bonamia exitiosa, Bonamia ostreae and Marteilia refringens were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, this studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms of reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected molluscs was not found, these were defined as reservoir. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir mollusc species during transport was collected from scientific literature. It was concluded that it is very likely to almost certain (90-100%) that M. mackini, P. marinus, B. exitiosa B. ostreae and M. refringens will remain infective at any possible transport condition. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild or at aquaculture establishments or through contaminated water supply can possibly transmit these pathogens. For transmission of M. refringens, the presence of an intermediate host, a copepod, is necessary.

Bonamia exitiosa in farmed native oysters Ostrea angasi in Australia: optimal epidemiological qPCR cut-point and clinical disease risk factors.

Bonamiosis has developed as a problem in Australian native oysters Ostrea angasi since the parasite Bonamia spp. was first detected in Port Phillip Bay, Victoria, in the early 1990s. At that time, large-scale mortalities in both farmed and wild oysters saw the demise of the pilot native oyster culture industry. More recent attempts to farm the species resulted in subclinical infections that progressed over time to clinical disease. The aim of this work was to establish what environmental factors result in the clinical manifestation of disease; determine the diagnostic sensitivity and diagnostic specificity of histopathological examination and a quantitative polymerase chain reaction (qPCR) test for the diagnosis of B. exitiosa infection in clinically diseased farmed native oysters; and calculate the optimal qPCR threshold cycle (CT) epidemiological cut-point for classification of positive and negative cases. After applying a range of stressors to tank-held oysters, results indicated a 58% increased risk (95% CI: 16%, 99%) of a Bonamia-infected oyster dying if the oyster was held at a higher temperature (p = 0.048). Starving and tumbling oysters, in isolation, was not significantly associated with clinical bonamiosis, but a Bonamia-infected oyster was at the greatest risk of death when increased water temperature was combined with both starvation and increased motion (p = 0.02; odds ratio = 3.47). The diagnostic sensitivity and specificity of the World Organisation for Animal Health qPCR protocol were calculated for increasing CT value cut-points from ≤25 to ≤40, with an optimal cut-point identified at ≤34.5 (specificity: 92.2; 95% posterior credible intervals [PCI]: 76.2, 99.8; Sensitivity: 93.5; 95% PCI: 84.7, 99.1).

Ephemeral detection of Bonamia exitiosa (Haplosporida) in adult and larval European flat oysters Ostrea edulis in the Solent, United Kingdom.

The haplosporidian parasite Bonamia exitiosa was detected using PCR in four adult and six larval brood samples of the European flat oyster Ostrea edulis from the Solent, UK. This represents the second reported detection of this parasite along the south coast of England. Adult oysters were collected and preserved from seabed populations or restoration broodstock cages between 2015 and 2018. The larvae within brooding adults sampled during 2017 and 2018 were also preserved. Molecular analysis of all samples was performed in 2019. The DNA of B. exitiosa was confirmed to be present within the gill tissue of one oyster within the Portsmouth wild fishery seabed population (n = 48), sampled in November 2015; the congeneric parasite Bonamia ostreae was not detected in this individual. This is the earliest record of B. exitiosa in the Solent. Concurrent presence of both B. ostreae and B. exitiosa, determined by DNA presence, was confirmed in the gill and heart tissue of three mature individuals from broodstock cages sampled in October 2017 (n = 99), two from a location on the River Hamble and one from the Camber Dock in Portsmouth Harbour. B. exitiosa was not detected in the November 2018 broodstock populations. A total of six larval broods were positive for B. exitiosa, with five also positive for B. ostreae. None of the brooding adults were positive for B. exitiosa suggesting that horizontal transmission from the surrounding environment to the brooding larvae is occurring. Further sampling of broodstock populations conducted by the Fish Health Inspectorate at the Centre for Environment, Fisheries and Aquaculture Science in June 2019 did not detect infection of O. edulis by B. exitiosa. These findings together suggest that the pathogen has not currently established in the area.

Infection dynamics of Bonamia exitiosa on intertidal Ostrea angasi farms.

Bonamia spp. cause epizootics in oysters worldwide. In southern Australia, Bonamia exitiosa Hine, Cochennac and Berthe, 2001 threatens aquaculture of Ostrea angasi Sowerby, 1871. Bonamia spp. infections can display strong seasonality, but seasonal dynamics of B. exitiosa-O. angasi are unknown. Ostrea angasi naïve to B. exitiosa infection were stocked onto farms in three growing regions, and B. exitiosa was monitored seasonally for one year. Environmental parameters we measured did not correlate with B. exitiosa prevalence or infection intensities. Extreme temperatures suggest O. angasi culture systems need development. Bonamia exitiosa prevalence increased over time. After three months, O. angasi had B. exitiosa prevalence of 0.08-0.4, and after one year, the prevalence was 0.57-0.88. At some sites, O. angasi had >0.5 B. exitiosa prevalence in >6 months, but at other sites, >9 months passed before prevalence was >0.5. Bonamia exitiosa infection intensities were low with no seasonal pattern but were affected by the interaction of site, season and oyster meat:shell ratio. Understanding infection and initiating a breeding programme for resistance would provide benefits for O. angasi industry expansion.

Rapid transmission of Bonamia exitiosa by cohabitation causes mortality in Ostrea angasi.

The haplosporidian Bonamia was first detected in Australian shellfish in 1991. Australian isolates in Ostrea angasi Sowerby, 1871 were identified as Bonamia exitiosa Hine, Cochennac and Berthe, 2001, which threatens development of an O. angasi aquaculture industry. European field data suggest that Bonamia ostreae Pichot, Comps, Tigé, Grizel and Rabouin, 1980 infections in Ostrea edulis Linnaeus, 1758 build slowly, but infection dynamics of B. exitiosa in O. angasi are unknown. We investigated B. exitiosa infection in O. angasi by cohabiting uninfected juvenile O. angasi with adults infected with B. exitiosa. Oysters were sampled at 10, 21 and 40 days after cohabitation, and B. exitiosa prevalence and intensity were assessed. Bonamia exitiosa rapidly infected and caused disease in O. angasi. Mortalities began at 12 days, with ˜50% mortality by day 21 and >85% mortality by day 40. Mortalities displayed pathology consistent with clinical B. exitiosa infection. Time to first infection is likely influenced by a combination of parasite infectivity, host exposure and host immune capacity. Host death is not required for transmission, but probably facilitates release of parasites from decaying tissue. Understanding B. exitiosa transmission informs design and interpretation of field studies and aids development of management strategies for oyster aquaculture.

PCR and histology identify new bivalve hosts of Apicomplexan-X (APX), a common parasite of the New Zealand flat oyster Ostrea chilensis.

Apicomplexan-X (APX) is a significant pathogen of the flat oyster Ostrea chilensis in New Zealand. The life cycle and host range of this species are poorly known, with only the zoite stage identified. Here, we report the use of molecular approaches and histology to confirm the presence of APX in samples of green-lipped mussels Perna canaliculus, Mediterranean mussels Mytilus galloprovincialis and hairy mussels Modiolus areolatus collected from widely distributed locations in New Zealand. The prevalence of APX infection estimated by PCR was 22.2% (n = 99) and 50% (n = 30) in cultured green-lipped mussels from Nelson and Coromandel, respectively; 0.8% (n = 258), 3.3% (n = 150) and 35.3% (n = 17) in wild Mediterranean mussels from Nelson, Foveaux Strait and Golden Bay, respectively; and 46.7% (n = 30) in wild hairy mussels from Foveaux Strait. Histology detected all cases of PCR that were positive with APX and appeared to be more sensitive. The prevalence of APX estimated by histology in green-lipped mussels from Coromandel was 60% versus 50% by PCR, and 4.3%, 10.7% and 52.9% by histology versus 0.8%, 3.3% and 35.3% by PCR in wild Mediterranean mussels from Nelson, Foveaux Strait and Golden Bay, respectively. The specific identity of the parasite found in mussels was determined by sequencing PCR products for a portion (676 bp) of the 18S rRNA gene; the resulting sequences were 99-100% similar to APX found in flat oysters. Phylogenetic analyses also confirmed that all isolates from green-lipped, Mediterranean and hairy mussels grouped with APX isolates previously identified from flat oysters. This study indicates the wide geographical distribution of APX and highlights the potentially multi-host specific distribution of the parasite in commercially important bivalve shellfish.

Proteomic profile of Ostrea edulis haemolymph in response to bonamiosis and identification of candidate proteins as resistance markers.

European flat oyster Ostrea edulis populations have suffered extensive mortalities caused by bonamiosis. The protozoan parasite Bonamia ostreae is largely responsible for this disease in Europe, while its congener B. exitiosa has been detected more recently in various European countries. Both of these intracellular parasites are able to survive and proliferate within haemocytes, the main cellular effectors of the immune system in molluscs. Two-dimensional electrophoresis was used to compare the haemolymph protein profile between Bonamia spp.-infected and non-infected oysters within 3 different stocks, a Galician stock of oysters selected for resistance against bonamiosis, a non-selected Galician stock and a selected Irish stock. Thirty-four proteins with a presumably relevant role in the oyster-Bonamia spp. interaction were identified; they were involved in major metabolic pathways, such as energy production, respiratory chain, oxidative stress, signal transduction, transcription, translation, protein degradation and cell defence. Furthermore, the haemolymph proteomic profiles of the non-infected oysters of the 2 Galician stocks were compared. As a result, 7 proteins representative of the non-infected Galician oysters selected for resistance against bonamiosis were identified; these 7 proteins could be considered as candidate markers of resistance to bonamiosis, which should be further assessed.

Whole-genome amplification: a useful approach to characterize new genes in unculturable protozoan parasites such as Bonamia exitiosa.

Bonamia exitiosa is an intracellular parasite (Haplosporidia) that has been associated with mass mortalities in oyster populations in the Southern hemisphere. This parasite was recently detected in the Northern hemisphere including Europe. Some representatives of the Bonamia genus have not been well categorized yet due to the lack of genomic information. In the present work, we have applied Whole-Genome Amplification (WGA) technique in order to characterize the actin gene in the unculturable protozoan B. exitiosa. This is the first protein coding gene described in this species. Molecular analysis revealed that B. exitiosa actin is more similar to Bonamia ostreae actin gene-1. Actin phylogeny placed the Bonamia sp. infected oysters in the same clade where the herein described B. exitiosa actin resolved, offering novel information about the classification of the genus. Our results showed that WGA methodology is a promising and valuable technique to be applied to unculturable protozoans whose genomic material is limited.

The haplosporidian Bonamia exitiosa is present in Australia, but the identity of the parasite described as Bonamia (formerly Mikrocytos) roughleyi is uncertain.

Protistan oyster parasites in the genus Bonamia have been observed in recent years infecting new hosts on five continents, with most of these parasites genetically similar to austral species Bonamia exitiosa and Bonamia roughleyi. Identification of the newly observed parasites as one or another of these described species has been complicated by the fact that B. exitiosa and B. roughleyi are phylogenetically indistinguishable at the small-subunit ribosomal DNA (SSU rDNA) level, with samples of B. roughleyi type material no longer available for genetic re-analyses using more informative internal transcribed spacer (ITS) region DNA sequences. To resolve this issue, we evaluated B. roughleyi in field collections of hosts Saccostrea glomerata and Ostrea angasi (as well as Crassostrea gigas) in New South Wales, Australia in 2006 and 2007, and re-analyzed histological samples from the original description of this parasite species using in situ hybridization. Despite (1) reports of the oyster disease putatively caused by B. roughleyi during the time of collections, (2) the observation of gross lesions characteristic of the disease, and (3) the observation of B. roughleyi cells in association with the lesions, we detected a Bonamia sp. by PCR in just 1/42 O. angasi (2.4%), and 1/608 S. glomerata (0.2%), the latter oyster of which is the type host. SSU rDNA sequences of the amplicons were nearly identical to those of B. exitiosa and B. roughleyi, and phylogenetic analysis of ITS region sequences placed them on a B. exitiosa clade. A Haplosporidium sp. sequence similar to that of H. costale was PCR-amplified from nearly half the S. glomerata and O. angasi, but no Haplosporidium sp. was observed histologically. Our inability to identify a Bonamia sp. sequence in association with the B. roughleyi observed histologically suggests that this parasite is not a Bonamia sp. at all, and should be regarded as B. roughleyi nomen dubium. We conclude that the Bonamia sp. that we and other investigators detected in southeastern Australian S. glomerata and O. angasi was B. exitiosa.