Development of a Protocol for Enrichment of HemogregarineInfected Fish Erythrocytes and Transmissible Cysts

Undergraduate #104
Discipline: Biological Sciences
Subcategory: Microbiology/Immunology/Virology

Shanan Emmanuel - University of the Virgin Islands
Co-Author(s): Jennilee Robinson, University of the Virgin Islands, Virgin Islands Andrew Campbell, Brown University, RI

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Caribbean dusky damselfish, Stegastes adustus, are infected with hemogregarine-like apicomplexan parasites. Little is known on the mode of transmission of these parasites from fish to fish. Specifically, it is unclear if the mode of transmission is via bloodfeeding crustaceans similar to Plasmodidae or by fecal-oral transmission like Sarcocystidae. We hypothesized that the hemogregarineis transmitted from host-to-host via cysts through the fecal-oral route, reminiscent of Toxoplasma infections in humans. To investigate this hypothesis we developed a protocol for isolation of cysts from fish fecal matter using Percoll step gradients, which separated the components of the fecal matter according to density. Preliminary results suggest that the parasite is transmitted through a fecal-oral pathway, because of the presence of sporulated and unporulated cysts. In addition, we developed a protocol for isolation and visualization of the infected erythrocytes from damselfish blood. Blood was also fractionated by differential centrifugation on Percoll gradients. Visualization of cells from fractionated blood employed stains including trypan blue, DAPI and pinacyanol chloride. Studies of the hemogregarine parasite can potentially serve as a good candidate model to study Toxoplasmosis in humans.

Funder Acknowledgement(s): 5T36GM101995-02; MARC 5T34GM008422

Faculty Advisor: Jennilee B. Robinson,