• Parasites, Hosts and Diseases
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• v.44 no.3
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• pp.197-207
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• 2006

This experiment focused on MAPK activation in host cell invasion and replication of T. gondii, as well as the expression of CC chemokines, MCP-1 and $MIP-1\alpha$, and enzyme, COX-2/prostaglandin $E_2(PGE_2)$ in infected cells via western blot, $[^3H]-uracil$ incorporation assay, ELISA and RT-PCR. The phosphorylation of ERK1/2 and p38 in infected HeLa cells was detected at 1 hr and/or 6 hr postinfection (PI). Tachyzoite proliferation was reduced by p38 or JNK MAPK inhibitors. MCP-1 secretion was enhanced in infected peritoneal macrophages at 6 hr PI. $MIP-1\alpha$ mRNA was increased in macrophages at 18 hr PI. MCP-1 and $MIP-1\alpha$ were reduced after treatment with inhibitors of ERK1/2 and JNK MAPKs. COX-2 mRNA gradually increased in infected RAW 264.7 cells and the secretion of COX-2 peaked at 6 hr PI. The inhibitor of JNK suppressed COX-2 expression. $PGE_2$ from infected RAW 264.7 cells was increased and synthesis was suppressed by PD98059, SB203580, and SP600125. In this study, the activation of p38, JNK and/or ERK1/2 MAPKs occurred during the invasion and proliferation of T. gondii tachyzoites in HeLa cells. Also, increased secretion and expression of MCP-1, $MIP-1\alpha$, COX-2 and $PGE_2$ were detected in infected macrophages, and appeared to occur via MAPK signaling pathways.

• Korean Journal of Veterinary Research
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• v.29 no.4
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• pp.567-575
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• 1989

This study was conducted to evaluate the possibility of application of a microenzyme-linked immunosorbent assay(micro-ELISA) for the serodiagnosis of specific toxoplasma antibodies in swine sera and this test was performed as a microplate system by coating the polystyrene plates with toxoplasma soluble antigen, incubated serially diluted sera, then added horse radish peroxidase labelled goat anti-swine IgG(r) conjugate followed by o-phenylenediamine as substrate. The color development by enzyme-substrate reaction was determined by the photometric reading [ELISA reader at 490nm (OD)] and visual reading. The soluble antigen was prepared from the tachyzoites in mouse peritoneal cavity. A total of 1,200 swine sera from pig slaughter-house and a total of 116 swine sera from pig breeding station (S-C farm) were tested for the detection of antibodies to Toxoplasma gondii. The results obtained were summarized as follows: 1. The optimal reactions of indirect ELISA for the test sera were determined by the dilution of antigen 1:256 and 1:3,200 of horse radish peroxidase conjugate [anti-swine IgG(r)]. 2. The specific togoplasma antibody(IgG) in pigs infected with Tp artificially were detected as the serum titers of 1:64 or 1:128 at one week postinfection. 3. Of a total of 1,200 swine sera from pig slaughter-house 505 samples of sera were detected as positive (42.1%) and of a total of 116 swine sera from S-C pig breeding station 68 samples of sera as positive (58.6%). 4. The specific antibody(IgG) detection rates against a total of 1,200 test sera from pig slaughter-house were not significant between male (43.1%) and female (40.7%). 5. The indirect ELISA was proved to be a sensitive and specific procedure for the serodiagnosis of swine toxoplasmosis and also evaluated as an effective screening test for the large scale of test samples in laboratory.

• Parasites, Hosts and Diseases
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• v.35 no.1
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• pp.55-62
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• 1997

The molecular weight 30 kDa membrane protein of Toxoplusma Sondii (Toxoplasma 30 kDa) apparently conserved in most strains of T. gondii and sera of infected hosts. The present study aimed to elucidate Toxoplasmc 30 kDa as a useful diagnotic antigen for serodiagnisis of toxoplasmosis by ELISA and for induction of protective immunity. Murine spleen cells immunized with the membrane antigen of T. gondii were fused with mouse Sp2/0-Ag 14 myeloma cells. Out of 8 clones selected, five were IgG2b, the others belonged to IgG 1 and IgG2a. The 30 kDa antigen was distributed mainly on the surface membrane of tachyzoites by indirect fluorescence method. Murine peritoneal macrophages which were activated by 30 kDa antigen produced more amounts of NO2 compared with crude antigen-treated group, however there were no significant differences in toxoplamacidal activity between the two groups. Higher specificity of Toxoplosma 30 kDa antigen was recognized for serodiagnosis of toxoplasmosis than the crude antigen. From these results, ToxopLasmo 30 kDa antigen enhances the cytotoxic effect of macrophages as well as a more reliable means for the serodiagnosis of toxoplasmosis by ELISA. Key words: Toxoplosma gondii, 30 kDa antigen (p30), mouse, serodiagnosis, macrophage, cytotoxicity.

• Korean Journal of Veterinary Research
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• v.38 no.1
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• pp.139-145
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• 1998

The Neospora sp. was isolated from the brain of 1 calf via continuous in vitro cultivation in Vero cell. Neospora tachyzoites were observed 45 days after inoculation of the homogenized brain suspension into the Vero cell. The isolated parasite (named tentatively as NCKB-1) was morphologically and ultrastructurally similar to the previously reported Neospora sp isolated in cattle (BPA-1, JPA-1). A comparison of the antigenic reactivity of cultivated tachyzoites with polyclonal antisera to Neospora caninum and Toxoplasma gondii confirmed that this protozoal isolate was similar to N caninum. This is the first report of successful isolation of Neospora sp from cattle in Korea.

• Parasites, Hosts and Diseases
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• v.46 no.1
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• pp.45-48
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• 2008

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 SOS-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.

• Parasites, Hosts and Diseases
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• v.55 no.5
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• pp.491-503
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• 2017

The effects of tyrosine kinase inhibitors (TKIs) were evaluated on growth inhibition of intracellular Toxoplasma gondii in host ARPE-19 cells. The number of tachyzoites per parasitophorous vacuolar membrane (PVM) was counted after treatment with TKIs. T. gondii protein expression was assessed by western blot. Immunofluorescence assay was performed using Programmed Cell Death 4 (PDCD4) and T. gondii GRA3 antibodies. The TKIs were divided into 3 groups; non-epidermal growth factor receptor (non-EGFR), anti-human EGFR 2 (anti-HER2), and anti-HER2/4 TKIs, respectively. Group I TKIs (nintedanib, AZD9291, and sunitinib) were unable to inhibit proliferation without destroying host cells. Group II TKIs (lapatinib, gefitinib, erlotinib, and AG1478) inhibited proliferation up to 98% equivalent to control pyrimethamine ($5{\mu}M$) at $20{\mu}M$ and higher, without affecting host cells. Group III TKIs (neratinib, dacomitinib, afatinib, and pelitinib) inhibited proliferation up to 98% equivalent to pyrimethamine at $1-5{\mu}M$, but host cells were destroyed at $10-20{\mu}M$. In Group I, TgHSP90 and SAG1 inhibitions were weak, and GRA3 expression was moderately inhibited. In Group II, TgHSP90 and SAG1 expressions seemed to be slightly enhanced, while GRA3 showed none to mild inhibition; however, AG1478 inhibited all proteins moderately. Protein expression was blocked in Group III, comparable to pyrimethamine. PDCD4 and GRA3 were well localized inside the nuclei in Group I, mildly disrupted in Group II, and were completely disrupted in Group III. This study suggests the possibility of a vital T. gondii TK having potential HER2/4 properties, thus anti-HER2/4 TKIs may inhibit intracellular parasite proliferation with minimal adverse effects on host cells.

• Parasites, Hosts and Diseases
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• v.34 no.3
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• pp.197-206
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• 1996

Water extract of Coix locrvmn seeds (Co-Ex) was separated into several components; dissolved with Tris-Cl buffer and the supernatant (WC 1), ammonium sulfate treatment supernatant (WC2) and the pellet nvc31,9AE column chromatography of WC 1 and the peak portions; WC4, WCS and WC6. Murine peritoneal macrophages in DMEM containing 10% heat-inactivated FCS were infected with tachyzoites of ToxopIQsmc gondii, RH strain, in uifo. By adding modulators such as Co-Ex, WC 1,2,3,4.5,6 and LPS or IFN-γ for 24 hrs . toxoplasmastatic activity of macrophages was examined in relation to nitrite production. Nitrite production of macrophages was enhanced especially in the series of WC2, WC1 and the combination sample (WC1 + WC2 + WC3) by order than other components or fractions (WC4, WC5, WC6) tested . Toxoplasmastatic actions such as percentage of the inacrophages infected by T. gonnii and fold increase of T gondii in macrophages showed retroverse relations with the amount of nitrite production; i.e. as nitric oxide (NO) increased the phagocytic index of macrophages and the fold increase of tachyzoites in macrophages decreased . Nitrite (NO-2) production was increased by adding IFN-γ in all cases together with enhancement of biostatic effects. Through the results obtained, it is speculated that some components other than the non-proteinous and defatted components in Coix lacrwmn seeds may contribute to activate macrophages through induction of NO for the biostatic activity.

• Korean Journal of Veterinary Research
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• v.38 no.4
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• pp.853-858
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• 1998

A case of repeated bovine abortion caused by Neospora caninum is described. The 3-year-old Holstein cow, which was seropositive to N caninum antibody by indirect immunofluoresent assay, aborted consecutively within a 10-month period. Two fetuses of 6- or 5-month of gestation were aborted in June 1996 and March 1997, respectively. Histologically, multifocal necrotizing encephalitis, nonsuppurative periportal hepatitis, myocarditis, and myositis were observed in both fetuses. Tachyzoites or tissue cysts detected in the brain, heart and liver were positive to N caninum but negative to Toxoplasma gondii by immunohistochemical method. The results of our study demonstrate that repeated abortion due to N caninum actually occurs in Korea. This is believed to the first report of repeated abortion associated with N caninum in Korea.

• Parasites, Hosts and Diseases
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• v.32 no.4
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• pp.249-258
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• 1994

Recently, the importance of toxoplasmosis is raised as a complication in immunosuppressed or AIDS patients. Our study focused on the identification of a variety of Toxoplasma antigens by immunoblotting. Rabbits and BALB/c mice were immunized with Toxoplosmo Iysate (RH strain) , frozen tachyzoites (RH strain) or cysts (Beverly and Fukaya strain) . Blood were collected from ear vein, heart or orbital plexus for detecting the serum antibody levels. For excretory-secretory (E.S) antigens, T gondii (RH) tachyzoite were cultured in CHL (Chinese hamster lung) cells with MEM containing of 5% FCS. After 72hrs, culture supernatant was collected. BALB/c mice were inoculated with RH tachyzoite intraperitoneally and peritoneal fluids were extracted three days later. E.S antigens were detected in culture supernatant and infected mouse peritoneal fluid by EITB. Serum IgG levels in rabbit were 1 :512 of 10 days after primary immunization, 1 : 2,048 of 10 days after secondary immunization, 1: 1,024 of 20 days after secondary immunization by IFAT, respectively. Serum IgG levels of immunized mice were 1:128 after 7 weeks. Tachyzoite antigens of the RH strain were detected 25 protein bands ranging 10 kDa-220 kDa of molecular weights with Coomassie blue stain. Toxoplcsma major antigens corresponding to n of 24 kDa, 27 kDa,30 kDa, 35 kDa, 38 kDa were recognized by IgG and IgM antibodies. Excretory-secretory antigens present in culture supernatant with M. W. of 20, 30 kDa and in infected mouse peritoneal fluid with M.W. of 33 (P30), 45 kDa. When RH tachyzoite antigen was probed with different mice sera immunized with 2 strains of T gondii, the IgG antibody bud of Fukaya and Beverly strain (8 week-serum) is identical to those of RH strain. It is considered that the 30 kDa polypeptide detected in excretory- secretory materials and Iysate was important major antigen of T gondii (RH).

• Parasites, Hosts and Diseases
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• v.35 no.4
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• pp.251-258
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• 1997

Tachyzoite antigens of Toxoplosnc gondii (RH) were partially purified by immunoaffinity chromatography. The cultivated ToxopLusmc in uiuo (mouse) and in nitro (Hep-2 cell) and peritoneal fluid of T. Bondii infected mice were collected for antigen analy- sis. Tachyzoite antigens collected from infected mouse showed positive bands of 76 kDa, 70 kDa,64 kDa, 53 kDa, 46 kDa, 44 kDa, 41 kDa, 35 kDa, 25 kDa, 18 kDa, and 13 kDa on immunoblot with anti-Toxoplcsmn rabbit sera, and those from infected Hep-2 cells revealed reactive bands of 70 kDa,64 kDa,53 kDa,35 kDa,28 kDa, and 13-10 kDa. After applying to an IgG-Sepharose column, two elusion peaks, E-1 and I-2 fractions, were obtained from both soluble antigen of T. gondii and the peritoneal fluid of infected mice, respectively. Immunoblots of soluble antigen with immunized rabbit sera revealed positive bands of 97 kDa, 63 kDa, 53 kDa and 35 kDa from I-1 fraction and 53 kDa and 35 kDa from I-2. In the case of the eluted peaks from mice peritoneal fluid, E-1 showed protein bands of 84 kDa,76 kDa,53 kDa and 29 kDa bands and 53 kDa and 45 kDa from I-2 on immunoblots. Serum IgG antibody titer of mice immunized with T gonnii tachyzoites was increased on 1 week after booster immunization when analysed by ELISA using crude antigen, while it was elevated on 3 weeks after booster immunization by ELISA using puri- fied antigen.