Recent reports of toxoplasmosis in marine mammals raise concern that cold-blooded marine animals are a potential source of Toxoplasma gondii infection. To examine the transmissibility of T. gondii to fish, we observed the development of T. gondii tachyzoites inoculated into oviduct epithelial cells of goldfish ( Carassius auratus ) microscopically in vitro. Further, the survival period of tachyzoites inoculated into goldfish muscle was bioassayed in mice and through PCR analysis. In cell cultures at 37 C, both RH and Beverley strains of T. gondii tachyzoites had penetrated into cells at 6 hr post inoculation, and were multiplying. In cell cultures at 33 C, many tachyzoites of both strains attached to the host cells, but no intracellular tachyzoites were observed at 24 hr post inoculation. In the T. gondii inoculated goldfish kept at 33 C, tachyzoite DNA was detected in the inoculated region on day 3, but not on day 7. When inoculated goldfish were kept at 37 C, live tachyzoites were seen at the inoculation site on day 3, but not on day 7. These results suggest that T. gondii does not persist in fish.

Because there has been no report of symptomatic Neospora caninum infection in humans, we examined the effect of human serum on the parasite's growth in either a bovine angioendothelial cell or Caco-2 cell culture in vitro and in immunocompromised mice in vivo. There was no difference in intracellular parasite numbers between cells incubated with human serum at 24 hr after challenge and those incubated with fetal bovine serum (FBS), which has no titer for the anti– N. caninum agglutination antibody test. Serum of sheep infected with N. caninum , which has the anti– N. caninum antibody, reduced the numbers of the intracellular parasite significantly. We also showed that there was no inhibitory effect on the intracellular multiplication of the parasite in cells incubated with human serum through incorporation of 3 H-uracil. CB-17 scid mice administered human serum daily and challenged with N. caninum died on day 20 or 22 after challenge, when large numbers of parasite clusters were found in the brain, oviduct, adrenal gland, lung, stomach, spleen, skeletal muscle, pancreas, and mesenteric lymph nodes. Scid mice administered FBS survived until the end of the experiment. These results suggest that adult human serum may have no inhibitory effect on the development of N. caninum in vitro and in vivo.

A soluble antigen isolated from Eimeria stiedai merozoites with a molecular mass of 49 kDa was detected in the bile of infected rabbits. Rabbits immunized with the antigen shed a lower number of oocysts than did nonimmunized rabbits postchallenge (p.c.). The immunized rabbits showed a marked and transient increase of alanine-aminotransferase (ALT) activity on day 8 p.c. The blood indocyanine green (ICG) clearance and r-glutamyltransferase (GGT) activity showed no change throughout the experiment. However, nonimmunized rabbits showed a gradual increase of ALT and GGT in the plasma and a delay of ICG p.c. Many merozoites were observed in the biliary ducts of the nonimmunized rabbits on day 8 p.c. using standard histology. In contrast, in the immunized rabbits, many inflammatory cells were observed around the biliary ducts, but there were few parasites in the tissue. These results suggest that the 49-kDa soluble protein antigen detected in the bile of the infected rabbits was a merozoite-specific antigen, and the immune reaction to the antigen may induce protective effects against the infection.

Neospora caninum was isolated from the brain of a naturally infected pregnant sheep by inoculation of immunodeficient mice with a homogenate of the brain tissue. The ewe showed no clinical signs. Tachyzoites were observed in the tissues of the nu/nu mice injected with the brain tissue homogenate and the diagnosis was confirmed by immunohistochemical staining with anti- N. caninum antibodies and by detecting N. caninum -specific DNA by polymerase chain reaction.

Neospora caninum tissue cysts were found in the brains of surgically delivered twin fetuses at 119 days of gestation. In the brains of both fetuses, there was an inflammatory reaction involving perivascular cuffings of mononuclear cells, glial nodules. The dam of these fetuses died because of metritis. Histopathological examination of the ewe revealed N. caninum tissue cysts and focal gliosis with mononuclear cell cuffings. A N. caninum -specific DNA fragment was detected in a brain homogenate of the ewe by the polymerase chain reaction method. This is the first report of N. caninum infection in twin ovine fetuses and in an adult sheep.