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Diseases of Aquatic Organisms

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DAO 35:213-219 (1999)  -  doi:10.3354/dao035213

Tetrazolium-based cytotoxicity assay to determine anti-protozoal activity against the scuticociliate Anophryoides haemophila

Alastair E. Cribb1, Beatrice Despres2, Richard J. Cawthorn2,*

Lobster Health Research Centre, 1Departments of Anatomy and Physiology, and 2Microbiology and Pathology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, C1A 4P3, Canada
*Addressee for correspondence. E-mail:

ABSTRACT: Anophryoides haemophila is a ciliated protozoan that causes 'bumper car' disease in American lobster Homarus americanus. Currently, there are no effective pharmaceuticals approved for use to treat the disease, which can be a significant source of mortality in lobster holding facilities. We have modified a tetrazolium-based cytotoxicity assay to determine the anti-protozoal activity of compounds towards A. haemophila. To obtain reproducible results, A. haemophila maintained in continuous culture were preferable to freshly isolated A. haemophila. It was also found that the ciliates must be replicating to produce reproducible results. Maximal anti-protozoal activity was evident after a 24 h incubation period. Oxytetracycline was found to be relatively inactive, while high concentrations (1 mM) of formaldehyde and sulphaquinoxaline were required to produce 50% cytotoxicity. In contrast, 100 µM lasalocid produced 92 ± 4% and 100 µM pyrimethamine produced 84 ± 6.5% cytotoxicity after 24 h. This objective assay provides a rapid means of screening compounds to identify those with potential in vivo activity against Anophryoides haemophila.

KEY WORDS: Drug · Anophryoids haemophila · Protozoa · Lobster · In vitro · Tetrazoliu

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