Low internal transcribed spacer rDNA variation in New Zealand Bonamia ostreae: evidence for a recent arrival.

Bonamia ostreae is a haplosporidian parasite of oysters that was first reported to occur in the Southern Hemisphere in 2015 in the New Zealand flat oyster Ostrea chilensis. Until that report, B. ostreae had been restricted to populations of O. edulis within the Northern Hemisphere. This large range extension raised questions regarding B. ostreae dispersal, including whether B. ostreae is a recent introduction and from where it originated. The whole 18S rRNA gene of New Zealand B. ostreae revealed 99.9-100% sequence homology to other published B. ostreae 18S rDNA sequences. Internal transcribed spacer (ITS) rDNA sequences (n = 29) were generated from New Zealand B. ostreae and compared to published B. ostreae sequences from 3 Northern Hemisphere sites: California, USA (n = 18), Maine, USA (n = 7), and the Netherlands (n = 6) to investigate intraspecific variation. Low ITS rDNA variation was observed from New Zealand B. ostreae isolates, and high levels of variation were observed from Northern Hemisphere B. ostreae sequences. We hypothesise that the low ITS rDNA diversity found in New Zealand B. ostreae is the result of a founder effect resulting from a single introduction from a limited number of propagules. The high level of ITS rDNA variation from the Northern Hemisphere prevented inferences of dispersal origins. New Zealand B. ostreae were genetically differentiated from all sites, and additional genetic data are required to better determine the origin of B. ostreae in New Zealand.

Pooled sample testing for Bonamia ostreae: A tale of two SYBR Green real-time PCR assays.

Pooled testing of samples is a common laboratory practice to increase efficiency and reduce expenses. We investigated the efficacy of 2 published SYBR Green real-time PCR assays when used to detect the haplosporidian parasite Bonamia ostreae in pooled samples of infected oyster tissue. Each PCR targets a different gene within the B. ostreae genome: the actin 1 gene or the 18S rRNA gene. Tissue homogenates (150 mg) of the New Zealand flat oyster Ostrea chilensis were spiked with ~1.5 × 103 purified B. ostreae cells to create experimental pools of 3, 5, and 10. Ten positive replicates of each pool size were assayed twice with each PCR and at 2 different amounts of DNA template. The PCR targeting the actin 1 gene was unable to reproducibly detect B. ostreae in any pool size. Conversely, the 18S rRNA gene PCR could reproducibly detect B. ostreae in pools of up to 5. Using a general linear model, there was a significant difference in the number of pools that correctly detected B. ostreae between each PCR ( p < 0.01) and each pool size ( p < 0.01). It is likely that the single copy actin 1 gene is more likely to be diluted and not detected by pooling than the multi-copy 18S rRNA gene. Our study highlights that validation data are necessary for pooled sample testing because detection efficacy may not be comparable to individual sample testing.

New pathological condition in cultured mulloway Argyrosomus japonicus: histopathological, ultrastructural and molecular studies.

Mulloway Argyrosomus japonicus is a native fish species in Western Australia, for which aquaculture production has recently been developed. A single cohort was stocked in a cage offshore at Geraldton, Western Australia, at a water depth of 6 m. Fish appeared healthy before stocking. Routine histological analysis was carried out from 10 mo post stocking and until completion of harvest (about 2.5 yr post stocking). No gross pathology was evident. Microscopically, however, granulomatous lesions were present in the kidneys of almost 100% of the fish examined. Enclosed in the granuloma was an aggregate of organisms, 4.2 to 5.4 µm in diameter. Kidney granulomas appeared as multi-focal aggregates. Granulomas at different stages of formation and finally fibrosing granulomas were observed. Granulomas also appeared infrequently in other organs: a few granulomas were found in the liver and spleen and a single granuloma in the heart of one fish. Transmission electron microscopy (TEM) revealed that the organism was composed of 2 cells, an outer cell enclosing an inner cell. The inner cell was surrounded by a double membrane and the outer cell by a single membrane. Cellular material, presumably of parasitic nature, surrounded the outer cell. The organism contained primitive mitochondria and abundant free ribosomes. Small subunit ribosomal DNA (SSU rDNA) sequence obtained by PCR revealed an 84% sequence identity with the myxosporean Latyspora scomberomori. Based on TEM and preliminary molecular results, we suggest that the organism is the extrasporogonic developmental stage of a myxozoan parasite, which failed to form spores in the mulloway host.

Comparison of three molecular methods for the detection of pilchard herpesvirus in archived paraffin-embedded tissue and frozen tissue.

Two epizootics affecting pilchards Sardinops sagax neopilchardus have been observed over their entire geographical range off the Australian coastline. The first occurred in 1995, involving high mortality (at least 10%) that devastated the pilchard population. The second occurred in 1998 and involved even higher mortality (70%). Both epizootics moved rapidly against the prevailing Leeuwin and East Australian currents from a defined point of origin. A herpesvirus, pilchard herpesvirus (PHV), was determined to be the cause of the epizootics, but the source of the virus remains unknown. In this research, in situ hybridization (ISH), polymerase chain reaction (PCR), and real-time PCR were compared for the detection of PHV in archived paraffin-embedded tissue and frozen tissue collected before, during, and after the 1995 epizootic. Results show that the conventional PCR failed to detect PHV in archived paraffin-embedded tissue, and that real-time PCR was the most sensitive of the 3 techniques and the best method for the detection of PHV.

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.

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.