In vitro and in vivo evaluation of quinine hydrochloride as a potential anti-protozoal for the eastern oyster parasite Perkinsus Marinus

Christina L. Panko

In light of lethal diseases, harvesting pressures, and anthropogenic impacts, the culture of the eastern oyster, Crassotrea virginica, began in order to enhance the shellfish industry. Perkinsus marinus is a particularly devastating protozoan that parasitizes the eastern oyster in estuaries along the eastern seaboard of the U.S. and the Gulf of Mexico. Despite the advent of oyster culture, P. marinus continues to pose problems. Typical parasite prevention or mitigation approaches under hatchery conditions often treat seawater and do not particularly treat infected oysters. Currently no anti-protozoan drug therapy exists in shellfish aquaculture to effectively treat P. marinus infected oysters. Benefits from successfully developing a chemical therapy to treat infected oysters could include; better quality gametes from select brood stock, prevention of disease transfers to grow-out areas, and P. marinus free oysters. Given the close taxonomic and phylogenetic proximity of P. marinus (close to Dinozoa) to another protozoan parasite, Plasmodium spp. (Apicomplexa), the antimalarial drug quinine was chosen for evaluation as a potential chemotherapeutic agent. This study focused on quinine's effect on the parasite, host immune cells (hemocytes), and infected host organisms. Under in vitro conditions, cultured P. marinus and oyster hemocyte viability was measured after acute challenges with various quinine HCl concentrations (10, 20, 25, and 50 ug/ml). A MTS/PMS dye reduction assay was utilized to determine the viability of cultured P. marinus meronts. Additionally, differences in susceptibility to quinine between six geographically distinct P. marinus isolates (ranging from Connecticut, New Jersey, Maryland, Virginia, Louisiana, and Texas) were determined. Hemocyte viability was measured with a neutral red dye uptake assay. Under in vivo conditions, P. marinus infected oysters were challenged with three quinine HCI concentrations (0.25, 2.5, and 25 mg/L). Oyster mortality was documented and when possible, tissue samples were taken for diagnosis of P. marinus infections over the course of the experiment. Condition index, P. marinus tissue infection intensity, and prevalence of P. marinus of remaining oysters were assessed after a three-week treatment period. The 50 ~g/ml (0.13 mM) quinine HCI concentration significantly inhibited 56% of P. marinus meronts after three hours. Additionally, the Connecticut and Texas cultures were most resistant to 50 ug/ml quinine HCI, while the Maryland isolate was most susceptible to 50 ~g/ml quinine HCL Although quinine HCI effectively inhibited P. marinus meronts under in vitro conditions, it negatively affected in vitro hemocyte viability (significantly decreased with 20, 25, and 501J.g/ml quinine HCI). There was 100% cumulative mortality after in vivo treatment with 25 and 2.5 mg/L quinine HCI within two weeks. Although the 25 mg/L concentration decreased P. marinus tissue infection intensity and prevalence, the concentration was toxic to the oysters. The lowest concentration of quinine HCI (0.25 mg/L) did not significantly decrease P. marinus intensity or prevalence in oyster tissue after the three week exposure. The in vivo results suggest that with this method of exposure, quinine HCI may have bioaccumulated to lethal concentrations in the oysters. A potential solution may be to lengthen the exposure with a very low concentration of quinine HCL The approach and methods of this research may serve as a complete evaluation of potential chemical therapies for aquaculture applications.

In vitro and in vivo evaluation of quinine hydrochloride as a potential anti-protozoal for the eastern oyster parasite Perkinsus Marinus

(1)