Progress in the development of shrimp cell cultures in Thailand.

Primary shrimp cell cultures were developed from lymphoid organ and ovaries of black tiger shrimp, Penaeus monodon, in double-strength Leibovitz's L-15 medium supplemented with 15% fetal bovine serum, 1% glucose, 5 g/L NaCl, 15% shrimp meat extract. The optimum conditions for primary culture in vitro were obtained in L-15 medium with an osmolality of approximately 730 +/- 10 mmol/kg, a temperature range of 25--28 degrees C and incubation in a normal atmosphere. However, basal medium supplemented with 0.01% cholesterol could enhance good growth and cells performance initiated from lymphoid organ. Both epithelial-like and fibroblastic- like cells were observed from those organs within 2 days incubation. Within 3 days, 80% confluent monolayers were obtained from the lymphoid organ while cultures from other tissues required 5 days. Cultures were maintained for at least 43 days. Only cells from lymphoid organ could be subcultured and confluent monolayers achieved within 10 days post-spilt. Healthy cultures of the lymphoid cells did not persist beyond the third passage. Application of these primary shrimp cell cultures for studying pathogenic viruses of shrimp in vitro will be discussed.

Light and electron microscope observations on presporogonic and sporogonic stages of Sphaerospora epinepheli (Myxosporea) in grouper (Epinephelus malabaricus).

Presporogonic (blood) stages of Sphaerospora epinepheli Supamattaya, Fischer-Scherl, Hoffmann, Boonyaratpalin, 1990 were observed in the circulating blood, sinus of kidney, glomerurar capillaries and liver arteries of grouper Epinephelus malabaricus. The earliest detectable stage was a primary cell with one secondary cell. After cell divisions, nine to 16 secondary cells were found in one primary cell. Ultrastructural examination revealed electron-dense bodies (118-145 nm) in the cytoplasm of primary cells. Sporogonic stages and spores were located in Bowman's space and in kidney tubule lumens. Electron micrographs revealed a similar pattern of spore development as described from other Sphaerospora spp. Kidneys infected with S. epinepheli showed highly vacuolated tubular epithelial cells and severely affected renal corpuscles.

Sphaerospora epinepheli n. sp. (Myxosporea: Sphaerosporidae) observed in grouper (Epinephelus malabaricus).

Sphaerospora epinepheli n. sp. is described from grouper, Epinephelus malabaricus, in cage-cultured and wild fish collected from both coastal lines of southern Thailand. Subspherical to spherical spores and mono- or disporous pseudoplasmodia were observed in the lumen of kidney tubules. Pseudoplasmodia were round to elongate, size range 15.6-22.9 microns (length) x 8.4-21.6 microns (width). Spores were 7.8-10.0 microns (length) x 12.3-14.5 microns (thickness), and 7.0-9.5 microns (width) with two spherical polar capsules of equal size measuring 2.9-4.4 microns in diameter and containing polar filaments with six or seven windings. Two uninucleate sporoplasms showed iodine vacuoles. Blood stages, similar to C-blood protozoans observed from freshwater fish in Europe, were found from peripheral blood smears of grouper. Ultrastructural studies of blood stages showed a similar structure to unidentified mobile protozoans from the blood of carp. Electron dense bodies were observed in the cytoplasm of the primary cell blood stages. Infected proximal-tubular epithelial cells showed highly vacuolated cytoplasm and pycnotic nuclei.