Development of Sarcocystis falcatula in cell cultures demonstrates that it is different from Sarcocystis neurona

The development of Sarcocystis falcatula merozoites in bovine turbinate (BT) cell cultures is described and compared with development of Sarcocystis neurona merozoites. Merozoites of S. falcatula entered BT cell cultures and increased in size until 3 days post-inoculation when the nucleus of some merozoites developed lobes. Developing schizonts present at 4 days contained a lobed nucleus or appeared multinucleate. A single mature schizont was observed 4 days p.i. Schizonts were numerous 5 and 6 days p.i. Merozoites were produced from blastophores on the schizont. S. neurona merozoites developed to mature schizonts by 3 days p.i. in BT cells and a residual body was often present. Transmission electron microscopy revealed that S. falcatula merozoites possessed more micronemes than did S. neurona merozoites. Our study demonstrates that S. falcatula and S. neurona are not the same parasite.

Immunogold-Silver Staining (IGSS) of (NCP and CP) BVDV Infected Subcellular Fraction Bands

Pre-embedding immunogold-silver (IGSS) techniques are useful to localize antigens in cell monolayers and agarose embedded cell suspensions for transmission electron microscopy. Procedural centrifugations, however, present a challenge when attempting to localize antigens in subcellular fractions. Using a Beckman Airfuge Ultracentrifuge to concentrate the subcellar fraction bands and resuspending the organelles in agarose simplifies IGSS processing and resin embedding procedures.Control bovine turbinate (BT), and BT cells infected with cytopathic (cp NADL) and non-cytopathic (ncp NY-1) strains of bovine viral diarrhea virus (BVDV) were fractioned according to Bienz et al (1992). Bands containing membrane vesicles (Fig 2) were collected and each fraction band was pelleted at 169,000g for 20 min using an A-95 fixed angle rotor in a Beckman Airfuge Ultracentrifuge. Each fraction pellet was resuspended in 50μl of 30% agarose, solidified, and trimmed to < lmm. IGSS procedures were carried out according to Nanoprobes, Inc., Stoney Brook, NY, and Hearne & Van Campen (1996).

Immunogold-Silver Staining (IGSS) of Agarose Embedded (NCP) Bvdv-Infected Cell Suspensions

The use of pre-embedding immunogold-silver staining (IGSS) in electron microscopy of cell culture monolayers has been reported by various authors and its usefulness to localize antigens has been documented. Embedding cell suspensions or cell fractions for IGSS can require time-consuming centrifugation steps. Embedding cell suspensions in agarose simplifies IGSS processing and embedding procedures.Bovine turbinate cells (BTs) were grown to confluence in 25cm3 culture flasks. BTs were infected with a non-cytopathic strain (ncp) of bovine viral diarrhea virus (BVDV) while control cells were sham inoculated. At 18 h pi, cells were detached with trypsin. All following procedures were carried out at RT. The cells were fixed for 30 min in 2.0% paraformaldehyde + 0.2% glutaraldehyde in 0.1 M. phosphate buffer, pH 7.4, and rinsed 3 times in 0.1 M phosphate buffer, pH 7.4 (each centrifuged for 10 min at 300g). The cells were resuspended in 1 ml of buffer, transferred to an eppendorf tube and centrifuged for 10 min at 300g. The excess buffer was removed, 1-2 drops of warm, molten 1.0% agarose added, and the mixture centrifuged for 1 min at 300g to pellet the cells to the tip of the tube. The solidified cell-agarose mixture was removed, cut into <0.5mm3pieces, and placed in phosphate buffer.