Partial 18S rRNA sequences of apicomplexan parasite 'X' (APX), associated with flat oysters Ostrea chilensis in New Zealand.

Apicomplexa is a large phylum of parasitic protists renowned for significant negative health impacts on humans and livestock worldwide. Despite the prevalence and negative impacts of apicomplexans across many animal groups, relatively little attention has been given to apicomplexan parasites of invertebrates, especially marine invertebrates. Previous work has reported an apicomplexan parasite 'X' (APX), a parasite that has been histologically and ultrastructurally identified from the commercially important flat oyster Ostrea chilensis in New Zealand. This apicomplexan may exacerbate host vulnerability to the infectious disease bonamiosis. In this study, we report 18S rRNA sequences amplified from APX-infected O. chilensis tissues. Phylogenetic analyses clearly established that the 18S sequences were of apicomplexan origin; however, their detailed relationship to known apicomplexan groups is less resolved. Two specific probes, designed from the putative APX 18S rRNA sequence, co-localised with APX cells in in situ hybridisations, further supporting our hypothesis that the 18S sequences were from APX. These sequences will facilitate the future development of inexpensive and sensitive molecular diagnostic tests for APX, thereby assisting research focussed on the biology and ecology of this organism and its role in morbidity and mortality of O. chilensis.

Bonamia ostreae in the New Zealand oyster Ostrea chilensis: a new host and geographic record for this haplosporidian parasite.

Previous reports of the haplosporidian parasite Bonamia ostreae have been restricted to the Northern Hemisphere, including Europe, and both eastern and western North America. This species is reported for the first time in New Zealand infecting the flat oyster Ostrea chilensis. Histological examination of 149 adult oysters identified 119 (79.9%) infected with Bonamia microcells. Bonamia generic PCR of several oysters followed by DNA sequencing of a 300 bp portion of the 18S rDNA gene produced a 100% match with that of B. ostreae. All DNA-sequenced products also produced a B. ostreae PCR-restriction fragment length polymorphism (PCR-RFLP) profile. Bonamia species-specific PCRs further detected single infections of B. exitiosa (2.7%), B. ostreae (40.3%), and concurrent infections (53.7%) with these 2 Bonamia species identifying overall a Bonamia prevalence of 96.6%. Detailed histological inspection revealed 2 microcell types. An infection identified by PCR as B. ostreae histologically presented small microcells (mean ± SE diameter = 1.28 ± 0.16 µm, range = 0.9-2 µm, n = 60) commonly with eccentric nuclei. A B. exitiosa infection exhibited larger microcells (mean ± SE diameter = 2.12 ± 0.27 µm, range = 1.5-4 µm, n = 60) with more concentric nuclei. Concurrent infections of both Bonamia species, as identified by PCR, exhibited both types of microcells. DNA barcoding of the B. ostreae-infected oyster host confirmed the identification as O. chilensis. A suite of other parasites that accompany O. chilensis are reported here for the first time in mixed infection with B. ostreae including apicomplexan X (76.5%), Microsporidium rapuae (0.7%) and Bucephalus longicornutus (30.2%).

Bonamiosis status in natural Ostrea puelchana beds in San Matías Gulf (Patagonia, Argentina), 14 years after an epizootic.

Between 1995 and 1996, Bonamia exitiosa caused an epizootic in San Matías Gulf, Argentina, that spread from a commercial culture site of Ostrea puelchana to natural beds located at the northeastern coast of the gulf. A mortality rate of 95% was registered in cultured oysters, and oysters from natural beds were also affected. The aims of this study were to assess the parasite prevalence in oyster beds and the demographic structure 14 yr after the epizootic. Two different oyster beds were studied during 2009 and 2010. Parasite prevalence was studied related to oyster aggregation, density, sex, and oyster size. Prevalence reached 35.3% at Las Grutas and 18.9% at Banco Reparo and was proportionally associated with density. Prevalence was also associated with the type of aggregation in Banco Reparo, where carrier oysters were more infected. Infection was independent of sex category, and infected oysters were larger than the non-infected ones. Oyster density decreased markedly compared to previous studies in both beds and mean sizes were lower, while prevalence doubled. Because of the persistence of the beds in this period, disease seems to control the population structure.

Gross signs and histopathology of Ostrea puelchana infected by a Bonamia exitiosa-like parasite (Haplosporidia).

Haplosporidian microcells belonging to the genus Bonamia parasitise various species of oysters around the world. In Argentina, Bonamia sp. was the causative agent of mass mortality among Ostrea puelchana cultured in San Antonio Bay (San Matías Gulf), and it was detected in natural beds inside San Matías Gulf. In order to describe the gross and histopathological signs caused by Bonamia sp. in O. puelchana, cultured and wild oysters were sampled and analysed by traditional techniques including heart imprints and histology. Cells of Bonamia sp. were observed in connective tissue, free or within haemocytes, in gills and around the digestive gland, stomach, intestine and gonad. Gross signs, histopathological alterations in O. puelchana, and Bonamia sp. cytological morphology resemble those reported for B. exitiosa. However, I propose to treat the Argentinean species as B. exitiosa-like until more molecular and ultrastructural studies are conducted to determine the correct taxonomy.

Ultrastructure of Bonamia sp. in Ostrea chilensis in Chile.

Oyster Ostrea chilensis samples were collected from Quihua Island, Chile, in December 2003 and February 2005, and examined in May 2004, and March, April and July 2005, for an ultrastructural comparison of the Chilean Bonamia sp. with other Bonamia spp. Only uni-nucleate stages were encountered, except in the July sample. The Chilean parasite differs from B. perspora in the apparent lack of a plasmodial stage and of sporulation. It resembles B. ostreae in size, the low number of mitochondrial profiles, and the prevalence and mean number of lipid droplets. It differs from B. ostreae in the greater prevalence of nuclear membrane-bound Golgi (NM-BG), associated haplosporogenesis, and smaller size of haplosporosomes. The Chilean Bonamia sp. resembles B. exitiosa in the number of haplosporosomes, prevalence of lipid droplets, anastomosing endoplasmic reticulum and NM-BG, presence of circles of smooth endoplasmic reticulum (sER), confronting cisternae (CC), and cylindrical CC (CCC). It also appears to have a similar developmental cycle to B. exitiosa with larger forms occurring in winter (August). The circles of sER, CC, and CCC have only been reported from B. exitiosa, and it appears that Chilean Bonamia sp. and B. exitiosa are more closely related than they are to B. perspora or B. ostreae. Similarities in ultrastructure and developmental stages between New Zealand and Chilean parasites suggest that the 2 species are related, and that the Chilean Bonamia sp. is either B. exitiosa, a sub-species of B. exitiosa, or a separate species closely related to B. exitiosa.