In vitro cultivation of the microsporidian: Enterocytozoon salmonis using a newly developed medium for salmonid lymphocytes

Techniques have been established for the continuous culture of a newly recognized salmonid microsporidian parasite Enterocytozoon salmonis in salmonid mononuclear leukocytes. Utilization of human recombinant interleukin-2 (HrIL-2) and polyclonal mitogens in the culture medium supported viability of salmonid mononuclear leukocytes which allowed proliferation of the intracellular microsporidian parasite. The microsporidian maintained in primary culture for 45 days was infective both in cultures of mononuclear leukocytes and in juvenile salmonids. Additional passages of the agents from primary cultures to new cultures of uninfected mononuclear leukocytes have allowed continuous in vitro propagation of E. salmonis. The parasites from these cultures retained developmental stages from early meronts to mature spores that were identical to those observed in naturally infected fish. Also, after cryopreservation in liquid nitrogen the parasites maintained their infectivity for juvenile chinook salmon and cell cultures of mononuclear leukocytes. The development of methods for the in vitro culture of E. salmonis has made possible investigation of the effect of this microsporidian on immune functions of its key target cell, the mononuclear leukocyte.