Isolation, culture, and optimal growth conditions for the shellfish protozoan parasite, Perkinsus olseni

Aquatic parasite cultures are a significant asset to improve our understanding of organism biology, transmission and disease progression, and evaluation of preventative and treatment measures. We adapted and optimized international standard methods for the isolation and culture of Perkinsus spp. for Perkinsus olseni. Perkinsus olseni cells were isolated from green-lipped mussels, Perna canaliculus, in Ray's fluid thioglycollate medium with incorporated antibiotics and grown in Dulbecco's Modified Eagle Medium-Ham's F-12 culture medium. Sequencing of the internal transcribed spacer region confirmed the culture was consistent with that previously reported for P. olseni (100% homology). Parasite morphology and life cycle stages were described and consistent with known stages of trophozoites, hypnospore, and zoosporangia and zoospores. A suspected additional reproductive mode which commonly occurred under poor nutritional conditions was comprised of trophozoite-like cells which developed into small motile zoospore-like cells. Mussel hemolymph supplement resulted in the rapid development of pre-zoosporangia into zoosporangia and the release of motile zoospores. Temperature had a significant effect on parasite propagation, which increased significantly above 20 °C and reached an optimum at 22 °C. At 35 °C Perkinsus proliferation was strongly reduced, suggesting that the upper thermal limit of P. olseni is 30 °C < T ≤ 35 °C. Cryopreserved cells recovered well under normal culture conditions. The optimised methods for the isolation and culture of P. olseni provide the foundation for subsequent in vivo and in vitro experimentation, and can advance our knowledge and management of this globally significant shellfish disease.