Yoshitaka Omata

Redescription of Neospora caninum and its differentiation from related coccidia

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Naturally-Occurring Neospora caninum Infection in an Adult Sheep and Her Twin Fetuses

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.

Isolation of Neospora caninum from the brain of a pregnant sheep.

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.

A survey of abortifacient infectious agents in livestock in Luzon, the Philippines, with emphasis on the situation in a cattle herd with abortion problems

In the Philippines, insufficient consideration has been given to the implementation of systematic control measures against major abortifacient infectious agents in livestock. To elucidate the epidemiology of abortifacient infectious agents in livestock, the prevalence of four abortifacient agents was assessed. Initially, a total of 96 cattle including 17 cows with history of abortion were examined in a herd in Luzon at the request of the farm owner. Six (35.3%) of the 17 aborting cows were found to be serologically positive for Neospora caninum (N. caninum), whereas the seroprevalence in non-aborting cows was 15.9% (10/63). Four of the 6 serologically positive aborting cows were also RT-PCR-positive for bovine viral diarrhea virus (BVDV). Two (12.5%) of the 16 bulls examined were also found to be infected with BVDV, suggesting a putative risk factor of transmission via semen. Based on sequence analysis, the isolates detected belong to BVDV type 1b group. Furthermore, an epidemiological survey of abortifacient infectious agents was conducted with various species of livestock from herds located in Luzon. Out of the 105 water buffalo samples collected, 4 (3.8%) were indicated positive to N. caninum, 2 (1.9%) to Toxoplasma gondii (T. gondii) and 2 (1.9%) to Trypanosoma evansi (T. evansi). The overall seroprevalence of N. caninum in goat and sheep were 23.6% (21/89) and 26.3% (10/38), respectively. BVDV was not detected in these herds. The findings of this exploratory study indicate a relationship between infection and bovine abortion and that a lager study is required to statistically confirm this relationship.

Transfer of antibodies to kittens from mother cats chronically infected with Toxoplasma gondii

By indirect immunofluorescence assay, anti-Toxoplasma gondii antibody levels were examined in fetuses and kittens born from chronically infected cats. Titer of anti-T. gondii IgG in sera of kittens born from infected cats was significantly high on the seventh day post-birth, and decreased to a serologically non-detectable level at 8-12 weeks post-birth under continuous suckling of maternal milk. Littermates nursed by a non-infected cat showed a faster rate of IgG antibody depletion. In sera of fetuses obtained from infected cats, anti-T. gondii IgG titer was lower than that of offspring born from infected cats. Anti-T. gondii IgM titer was non-detectable in sera of all kittens and fetuses. Kittens born from infected cats inoculated with T. gondii oocysts on Day 35 after birth shed oocysts and showed a transient increase of anti-T. gondii IgM titer. Findings in this study suggest that anti-T. gondii antibody IgG in kittens is transferred mainly via colostrum and the kittens that receive maternal anti-T. gondii antibodies develop inadequate resistance to T. gondii infection.

Antibodies to Brucella spp. in Pacific Bottlenose Dolphins from the Solomon Islands

Brucella spp. have been recently isolated from a variety of marine mammals. Serum samples from 58 Pacific bottlenose dolphins (Tursipa aduncus) from the Solomon Islands were tested for antibodies to Brucella spp. by the tube agglutination test (TAT), enzymed-linked immunosorbent assay (ELISA), and immunoblotting. Anti-Brucella spp. antibodies were detected by TAT and ELISA in 31 and 40 of 58 samples, respectively. These results suggest that Pacific bottlenose dolphins from the Solomon Islands are infected with Brucella spp. or a Brucella-like organism.

Correlation between antibody levels in Toxoplasma gondii infected pigs and pathogenicity of the isolated parasite

Sera and diaphragm muscle tissues were obtained from 109 commercial pigs between September 1991 and May 1992 from the slaughterhouse at La Plata, Provincia Buenos Aires, Argentina. Anti-Toxoplasma gondii IgG antibody reactivity to T. gondii antigens were assayed using sera by indirect immunofluorescence assay and immunoblotting technique. Anti-T. gondii IgG titers at serum dilutions of 1:1024 and higher were noted in 11.0% of the tested sera, and at dilutions of 1:16 and lower in 36.7% of the serum samples. Using mouse inoculation test, T. gondii was isolated from 14 pig diaphragm samples. Of five samples derived from pigs with antibodies at dilutions of 1:1024 and higher, four contained trophozoites which, when inoculated into mice intraperitoneally, killed all recipient hosts within 15 days post inoculation. Parasites detected in seven out of eight samples from pigs with antibodies at serum dilutions of 1:64 and lower formed cysts in the brain, and mice survived longer than 13 days post inoculation. Immunoblotting demonstrated antibody reactivity in pig sera samples with relatively high titers for parasite antigens. Results of the present study suggest that antibody production in infected pigs is apparently dependent on the pathogenicity of the parasite strain.

Acute restraint stress enhances calcium mobilization and proliferative response in splenic lymphocytes from mice

Calcium (Ca2+ ) plays an essential role in lymphocyte activation and maturation. Acute and chronic stress has been shown to modulate the lymphocyte immune response; but the relationship between cytosolic free Ca2+ concentration ([Ca2+ ]i) and the immune response in lymphocytes following exposure to stress has not been examined. In the present study, we investigated the effects of acute restraint stress on [Ca2+ ]i and the proliferation of splenic lymphocytes from mice. We observed that 2 h of restraint significantly increased plasma corticosterone levels in mice. On examining [Ca2+ ]i and the proliferation ex vivo of splenic lymphocytes isolated from restraint-stressed mice using fura-2 and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, respectively, we found that acute restraint stress caused a significant increase in resting [Ca2+ ]i and significantly enhanced the ability of concanavalin A (Con A; a T-cell-selective mitogen) to increase [Ca2+ ]i but not that of lipopolysaccharide (LPS; a B-cell-selective mitogen). In addition, acute restraint stress significantly enhanced Con A-stimulated but not LPS-stimulated lymphocyte proliferation. Overall, there was a positive correlation between [Ca2+ ]i and T-cell proliferation following acute restraint stress. The enhancements of [Ca2+ ]i and T-cell proliferation were completely suppressed by verapamil (a Ca2+ channel blocker). These results suggest that acute restraint stress enhances Con A-stimulated T-cell proliferation by increasing [Ca2+ ]i via stimulation of Ca2+ entry.

GROWTH INHIBITORY EFFECT OF BOVINE LACTOFERRIN ON TOXOPLASMA GONDII TACHYZOITES IN MURINE MACROPHAGES : ROLE OF RADICAL OXYGEN AND INORGANIC NITROGEN OXIDE IN TOXOPLASMA GROWTH-INHIBITORY ACTIVITY

To study the effector pathway of Toxoplasma growth-inhibitory activity induced by lactoferrin in murine macrophage, the role of reactive oxygen intermediates (O2-) and inorganic nitric oxide (NO) was examined. Production of O2- was diminished in cultures of macrophages supplemented with lactoferrin and the effect of lactoferrin was dose and time dependent. Production of NO was enhanced in cultures of macrophages supplemented with interferon-gamma, but not with lactoferrin. These findings suggest that this Toxoplasma growth-inhibitory activity induced by lactoferrin in macrophages is not mediated by O2- or NO molecules. A competitive inhibitor of the L-arginine dependent effector pathway, NG-monomethyl-L-arginine (NG MMA), virtually abolished the inhibitory effects induced by interferon-gamma. Similarly, the inhibitory activity induced by lactoferrin was also diminished in cultures supplemented with NG MMA. From these findings, it appears that the Toxoplasma growth-inhibitory activity induced by lactoferrin in macrophages may be mediated by an L-arginine-dependent effector pathway that does not involve NO production.

Isolation of coccidian enteroepithelial stages of Toxoplasma gondii from the intestinal mucosa of cats by Percoll density-gradient centrifugation

Abstract A method for isolation of enteroepithelial stages of Toxoplasma gondii from the intestinal mucosa of experimentally infected cats was developed using Percoll density-gradient centrifugation. Gamonts and merozoites were obtained essentially free of host-cell debris. A recovery rate of nearly 30% of the parasites in the original preparations was obtained by this method. Merozoites were separated from gamonts by filtration through a 3-μm polycarbonate filter.