Immune Responses of Mice Intraduodenally Infected with Toxoplasma gondii KI-1 Tachyzoites

Toxoplasma gondii Korean isolate (KI-1) tachyzoites were inoculated intraduodenally to BALB/c mice using a silicon tube, and the course of infection and immune responses of mice were studied. Whereas control mice, that were infected intraperitoneally, died within day 7 post-infection (PI), the intraduodenally infected mice survived until day 9 PI (infection with 1x10(5) tachyzoites) or day 11 PI (with 1x10(6) tachyzoites). Based on histopathologic (Giemsa stain) and PCR (B1 gene) studies, it was suggested that tachyzoites, after entering the small intestine, invaded into endothelial cells, divided there, and propagated to other organs. PCR appeared to be more sensitive than histopathology to detect infected organs and tissues. The organisms spread over multiple organs by day 6 PI. However, proliferative responses of splenocytes and mesenteric lymph node (MLN) cells in response to con A or Toxoplasma lysate antigen decreased significantly, suggesting immunosuppression. Splenic CD4+ and CD8+ T-lymphocytes showed decreases in number until day 9 PI, whereas IFN-gamma and IL-10 decreased slightly at day 6 PI and returned to normal levels by day 9 PI. No TNF-alpha was detected throughout the experimental period. The results showed that intraduodenal infection with KI-1 tachyzoites was successful but did not elicit significant mucosal immunity in mice and allowed dissemination of T. gondii organisms to systemic organs. The immunosuppression of mice included reduced lymphoproliferative responses to splenocytes and MLN cells to mitogen and low production of cytokines, such as IFN-gamma, TNF-alpha, and IL-10, in response to T. gondii infection.

Proteomic Analysis of Toxoplasma gondii KI-1 Tachyzoites

We studied on the proteomic characteristics of Toxoplasma gondii KI-1 tachyzoites which were originally isolated from a Korean patient, and compared with those of the well-known virulent RH strain using 2-dimensional electrophoresis (2-DE), mass spectrometry, and quantitative real-time PCR. Two-dimensional separation of the total proteins isolated from KI-1 tachyzoites revealed up to 150 spots, of which 121 were consistent with those of RH tachyzoites. Of the remaining 29 spots, 14 showed greater than 5-fold difference in density between the KI-1 and RH tachyzoites at a pH of 5.0-8.0. Among the 14 spots, 5 from the KI-1 isolate and 7 from the RH strain were identified using MALDI-TOF mass spectrometry and database searches. The spots from the KI-1 tachyzoites were dense granule proteins (GRA 2, 3, 6, and 7), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGRPTase), and uracil phosphoribosyltransferase (UPRTase). The spots from the RH strain were surface antigen 1 (SAG 1), L-lactate dehydrogenase (LDH), actin, chorismate synthase, peroximal catalase, hexokinase, bifunctional dihydrofolate reductase-thymidylate synthase (DHTR-TS), and nucleoside-triphosphatases (NTPases). Quantitative real-time PCR supported our mass spectrometric results by showing the elevated expression of the genes encoding GRA 2, 3, and 6 and UPRTase in the KI-1 tachyzoites and those encoding GRA 7, SAG 1, NTPase, and chorismate synthase in the RH tachyzoites. These observations demonstrate that the protein compositions of KI-1 and RH tachyzoites are similar but differential protein expression is involved in virulence.

Suppression of CD4+ T-Cells in the Spleen of Mice Infected with Toxoplasma gondii KI-1 Tachyzoites

Toxoplasma gondii KI-1, a recent new isolate from Korea, shows similar pathogenicity and infectivity to mice compared to the virulent RH strain. To understand characteristics of host immunity, including immune enhancement or suppression, we investigated proliferative responses and phenotypes of spleen cells. In addition, kinetics of IFN-gamma, a Th1 cytokine, was examined in BALB/c mice up to day 6 post-infection (PI). Intraperitoneal injection of mice with 103 KI-1 tachyzoites induced significant decreases (P < 0.05) in proliferative responses of spleen cells. This occurred at days 2-6 PI even when concanavalin A (con A) was added and when stimulated with KI-1 antigen, suggesting suppression of the immunity. CD4+ T-cells decreased markedly at day 2 PI (P < 0.05), whereas CD8+ T-cells, NK cells, and macrophages did not show significant changes, except a slight, but significant, increase of CD8+ T-cells at day 6 PI. The capacity of splenocytes to produce IFN-gamma by con A stimulation dropped significantly at days 2-6 PI. These results demonstrate that intraperitoneal injection of KI-1 tachyzoites can induce immunosuppression during the early stage of infection, as revealed by the decrease of CD4+ T-cells and IFN-gamma.

Evaluation of the Korean Isolate-1 Tachyzoite Antigen for Serodiagnosis of Toxoplasmosis

To evaluate the usefulness of the Korean Isolate-1 (KI-1) antigen for serodiagnosis of toxoplasmosis, antigen profiles of KI-1 tachyzoites were analyzed in comparison with RH tachyzoites by SDS-PAGE and immunoblotting. ELISA was performed on latex agglutination (LA)-positive and negative serum samples using KI-1 and RH antigens. Immunoblotting of the KI-1 antigen showed multiple antigen bands with molecular sizes of 22-105 kDa. Among them, 1 and 6 common bands were noted against a KI-1-infected and a RH-infected human serum, respectively, which represented differences in antigenic profiles between KI-1 and RH tachyzoites. However, all 9 LA-positive human sera were found positive by ELISA, and all 12 LA-negative sera were negative by ELISA; the correlation between the ELISA titers and LA titers was high (r = 0.749). Our results suggest that tachyzoites of KI-1 may be useful for serodiagnosis of human toxoplasmosis.

Evaluation of the Korean Isolate-1 Tachyzoite Antigen for Serodiagnosis of Toxoplasmosis

To evaluate the usefulness of the Korean Isolate-1 (KI-1) antigen for serodiagnosis of toxoplasmosis, antigen profiles of KI-1 tachyzoites were analyzed in comparison with RH tachyzoites by SDS-PAGE and immunoblotting. ELISA was performed on latex agglutination (LA)-positive and negative serum samples using KI-1 and RH antigens. Immunoblotting of the KI-1 antigen showed multiple antigen bands with molecular sizes of 22-105 kDa. Among them, 1 and 6 common bands were noted against a KI-1-infected and a RH-infected human serum, respectively, which represented differences in antigenic profiles between KI-1 and RH tachyzoites. However, all 9 LA-positive human sera were found positive by ELISA, and all 12 LA-negative sera were negative by ELISA; the correlation between the ELISA titers and LA titers was high (r = 0.749). Our results suggest that tachyzoites of KI-1 may be useful for serodiagnosis of human toxoplasmosis.

Genotyping of a Korean isolate of Toxoplasma gondii by multilocus PCR-RFLP and microsatellite analysis

Although the Korean isolate KI-1 of Toxoplasma gondii has been considered to be a virulent type I lineage because of its virulent clinical manifestations, its genotype is unclear. In the present study, genotyping of the KI-1 was performed by multilocus PCR-RFLP and microsatellite sequencing. For 9 genetic markers (c22-8, c29-2, L358, PK1, SAG2, SAG3, GRA6, BTUB, and Apico), the KI-1 and RH strains exhibited typical PCR-RFLP patterns identical to the type I strains. DNA sequencing of tandem repeats in 5 microsatellite markers (B17, B18, TUB2, W35, and TgM-A) of the KI-1 also revealed patterns characteristic of the type I. These results provide strong genetic evidence that KI-1 is a type I lineage of T. gondii.

Genetic characteristics of the Korean isolate KI-1 of Toxoplasma gondii

Toxoplasma gondii tachyzoites were isolated from an ocular patient in the Republic of Korea and maintained in the laboratory (designated KI-1). In the present study, its genotype was determined by analyzing dense granule antigen 6 (GRA6) gene and surface antigen 2 (SAG2) gene as typing markers. Digestion of the amplification products of GRA6 and of the 5' and 3' ends of SAG2, respectively, with Mse I, Sau3A I, and Hha I, revealed that KI-1 is included in the genotype I, which includes the worldwide virulent RH strain. In addition, when the whole sequences of the coding regions of SAG1, rhoptry antigen 1 (ROP1), and GRA8 genes of KI-1 were compared with those of RH, minor nucleotide polymorphisms and amino acid substitutions were identified. These results show that KI-1 is a new geographical strain of T. gondii that can be included in the genotype I.

Laboratory passage and characterization of an isolate of Toxoplasma gondii from an ocular patient in Korea

Toxoplasma gondii tachyzoites were isolated from the blood of an ocular patient, and have been successfully passaged in the laboratory, for over a year, by peritoneal inoculation in mice. The isolated parasite was designated the Korean Isolate-1 (KI-1) and its characteristics were compared with those of the RH strain, a wellknown virulent strain originating from a child who suffered from encephalitis. The morphology, pathogenicity, infectivity and cell culture characteristics of the KI-1 were similar to those of the RH strain. Both RH and KI-1 antigens were detected by an anti-T. gondii monoclonal antibody (mAb), Tg563, against the major surface protein SAG1 (30 kDa), whereas no reaction was observed against an anti-Neospora caninum mAb, 12B4. The KI-1 was confirmed as an isolate of T. gondii. A long-term laboratory maintenance and characterization of a local T. gondii isolate is reported for the first time in the Republic of Korea.