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The identification and distribution of an intracellular ciliate in pearl oysters, Pinctada maxima (Jameson 1901)

Spiers, Zoe (2008) The identification and distribution of an intracellular ciliate in pearl oysters, Pinctada maxima (Jameson 1901). PhD thesis, Murdoch University.

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In October 2001, a new intracellular parasite was detected in Western Australian pearl oysters, Pinctada maxima. This ciliated parasite had not previously been seen in the area despite surveillance over preceding years. A study was performed to identify its characteristics and distribution. The information provided by this study could thereby assist the Pearl Producers Association and Government bodies in determining the industry’s response to infections.
The morphology of the intracellular ciliate of pearl oysters was investigated using light microscopy, revealing organisms that were approximately 5 x 11 μm in size, with 9 rows of cilia and a lobulated nucleus. The ciliates were located within the apical portion of the cytoplasm of digestive epithelial cells of the digestive gland. Investigations into the ultrastructure of the ciliate using transmission electron microscopy (TEM) supported the findings made using histology.
On initial morphological examination, a similarity between the pearl oyster parasite and a ciliate found in Canadian mussels was identified. Samples of Canadian mussels infected with their intracellular ciliate were analysed using light microscopy and TEM. This identified almost identical morphological characteristics to the Western Australian ciliate of pearl oysters, with a slight difference in size that could be attributed to fixation and processing.
The successful culture of adductor muscle, mantle tissue and digestive gland derived cells from pearl oysters was developed and maintained in vitro for up to 75 days, using a number of varying cell culture media. This was attempted in order to provide an ability to study the live intracellular organisms in the laboratory. Contamination and slow growth were the main issues affecting the viability of the technique.
An investigation into the pathology associated with the presence of the ciliate in the pearl oysters was performed. A positive correlation between the presence of the ciliate and an inflammatory response of the digestive gland was displayed using statistical techniques. Pathological changes to the infected cells were demonstrated using TEM, indicating disrupted cytoplasm, vacuolation and nuclear degeneration.
Field trials placing naïve spat in pearl farm leases that had prior ciliate infections were performed. No intracellular ciliates were detected in P. maxima. Other bivalve species from the same regions were examined histologically for health and parasites. With a total of 345 bivalves surveyed during 2005 to 2007, from 8 geographical locations and over 11 species of bivalve included, one oyster contained the intracellular parasite. This oyster was a 20 mm male bastard shell (Pinctada sp.) from Gales Bay, sampled in October 2006. One protozoal parasite was identified in a novel host bivalve species, and a previously undescribed single celled organism was also discovered.
An experimental design for a cross infection trial, should the ciliate become available, was completed. The sensitivity of histology to detect ciliates was also examined, revealing its sensitivity as a diagnostic tool in at risk populations was low (38%) to moderate (50%).
The historical distribution of the ciliate was examined using archival records, indicating that the highest prevalences occurred in the warmer months of October to February from 2001 to 2006. This also corresponded to the months of reduced rainfall.
An attempt at designing a PCR test to determine the molecular characteristics of the ciliate was performed. Formalin fixed, paraffin embedded oyster DNA was successfully extracted, amplified and sequenced, however isolation of ciliate DNA remained elusive, and may have been out-competed with host DNA. In situ hybridisation displayed positive staining with a probe designed from the Ciliophora 16s ssu gene.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Biomedical Sciences
Supervisor(s): Raidal, Shane, Jones, Brian and O'Hara, A. J.
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