• Parasites, Hosts and Diseases
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• 제57권5호
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• pp.537-541
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• 2019

Toxoplasma gondii infection induces parasite infiltration and apoptosis in the spleen. However, dose-dependent parasite infiltration, apoptosis, body weight alternations and survival in mice remain largely unknown. In this study, mice were intraperitoneally infected with 10, 30 or 100 tachyzoites of T. gondii, respectively. Parasite infiltration and apoptosis in the spleen were analyzed on days 3, 7, and 9 post-infection by immunohistochemistry and flow cytometry. Significantly higher levels of T. gondii infiltration and apoptosis in the spleen were found in 30 and 100 tachyzoites infected mice compared to 10 tachyzoites infected mice on days 7 and 9 post-infection. Although 30 and 100 tachyzoites infected mice showed significant body weight loss compared to 10 tachyzoites infected mice, all of the 100, 30, and 10 tachyzoites infected mice died by days 12, 15, and 17, each respectively. Interestingly, T. gondii infiltration in 10 tachyzoites infected mice were limited to capsule area of the spleen on day 9 post-infection. Several areas of parasite infiltrations were found in the 30 tachyzoites infected mice, where noticeable levels of splenic capsule de-adhesion occurred. These results indicated that parasite infiltration and apoptosis in the spleen, as well as body weight loss (survival) are closely correlated with infection dosage. The level of T. gondii infiltration and apoptosis in the spleen and splenic de-adhesion were dependent on the parasite dose.

• Parasites, Hosts and Diseases
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• 제48권3호
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• pp.195-201
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• 2010

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 tachyzoties 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 nucleosidetriphosphatases (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.

• Parasites, Hosts and Diseases
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• 제42권4호
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• pp.185-193
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• 2004

Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of $Ca^{2+}$ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and $Ca^{2+}$ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the $Ca^{2+}$ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular $Ca^{2+}$ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.

• 대한약침학회지
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• 제19권1호
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• pp.70-73
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• 2016

Objectives: This study was performed to determine the lethal and the inhibitory effects of ethanol extracts of Achillea millefolium (A. millefolium) and Hypericum perforatum (H. perforatum) on Toxoplasma gondii (T. gondii) RH strain tachyzoites in vitro. Methods: The tachyzoites were treated with concentrations of 10, 50, and 100 mg/mL of A. millefolium and H. perforatum extracts within 10, 30, and 45 minutes in the wells. The mortality rates of tachyzoites treated with extracts were determined by using alkaline methylene blue staining. Also, the tachyzoites in cell cultures were treated with concentrations of 50, 100, and 200 mg/mL of these extracts. The cell viability, inhibition concentration ($IC_{50}$), and selectivity were determined from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Results: In the cell-free in vitro study, all of tachyzoites were killed at concentrations of 100 mg/mL of both extracts while at concentration 10 mg/mL, the mortality was 4.53% - 5.31%. In the cell culture study, the values of the effective concentration ($EC_{50}$) were 215 and $153{\mu}g/mL$ and the selectivities were 0.73 and 0.69 for the A. millefolium and the H. perforatum extracts, respectively. Conclusion: We conclude that neither extracts has any significant effect on the tachyzoites of T. gondii in cell cultures.

• 한국동물위생학회지
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• 제22권2호
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• pp.129-134
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• 1999

A preservation test of Toxoplasma gondii tachyzoites for considerable time was tried to obtain simple and economical methods using various suspending media at $4^{\circ}C$ rather than serial passage of the parasite in mice. The preservation period was a term that the tachyzoites were detected from the peritoneal fluid of mice after inoculation of 2$\times$$10^5$ organisms preserved according to the lapse of time. The numbers of tachyzoite per 1\textrm{mm}^3$ of the peritoneal fluid with 2$m\ell$ of the saline solution taken in 4days after inoculation were presented as percentage in proportion to the control. The numbers. of tachyzoite per 1\textrm{mm}^3$ of the peritoneal fluid of the control were consisted of the average number of the tachyzoites of 10 mice inoculated with 2$\times$$10^5$ organisms by serial mouse passage. The tachyzoites could be preserved for 26 days in the suspending medium of saline solution at $4^{\circ}C$ Ringer's solution for 18 days, Hank's solution for 28 days, and egg-glycerine solution for 50 days.

• Journal of Microbiology and Biotechnology
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• 제12권4호
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• pp.628-634
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• 2002

A microfilament-based motility in Toxoplasma gondii (T. gondii) Is involved in host cell invasion, yet the exact mechanism has not yet been determined. Accordingly, the current study examined the localization of actin and tubulin in T gondii using immunofluorescent (IF) and immunogold staining for electron microscopy. Indirect immunofluorescence (IF) staining using anti-actin and anti-tubulin monoclonal antibodies (mAbs) revealed localization of fluorescence on the entire surface of the tachyzoites. The actin in T. gondii was observed by immunogold staining, and the gold particles were seen on the surface, especially at the anterior end and in the cytoplasm of the parasite. However, there were no gold particles in the nucleus, rhoptries, and dense granules. The tubulin in T gondii was located on the surface and in the cytoplasm of the tachyzoites in the extracellular parasite, compared with anterior part of tachyzoites in the intracellular parasite. The antigens of T gondii recognized by anti-actin mAb were 107 kDa, 50 kDa, 48 kDa, and 40 kDa proteins, while those recognized by anti-tubulin mAb were 56 kDa, 52 kDa, and 34 kDa proteins. Tachyzoites of T gondii pretreated with the actin inhibitor, cytochalasin D (20 $\mu\textrm{g}$/ml), and tubulin inhibitor, colchicine (2$\times$10$\^$-6/ M), for 30 min at 37$\^{C}$ were used to infect the isolated mouse macrophages (tachyzo ites:macrophage=2:1). Pretreatment with the inhibitors resulted in lower multiplication of tachyzoites within the macrophages than in the untreated group 18 h post infection (p<0.05). Therefore, the present results suggest that actin and tubulin appear to be involved in the invasion of and multiplication in host cells.

• Parasites, Hosts and Diseases
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• 제39권3호
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• pp.233-240
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• 2001

Although some reports have been published on the protective effect of antibodies to Toxoplasma gondii surface membrane proteins, few address the inhibitory activity of antibodies to dense granular proteins (GRA proteins) . Therefore, we performed a series of experiments to evaluate the inhibitory effects of monoclonal antibodies (mAbs) to GRA proteins (GRA2, 28 kDa; GRA6, 32 kDa) and surface membrane protein (SAGI, 30 kDa) on the invasion of T. gondii tachyzoites. Passive immunization of mice with one of three mAbs following challenge with a lethal dose of tachyzoites significantly increased survival compared with results for mice treated with control ascites. The survival times of mice challenged with tachyzoties pretreated with anti-GRA6 or anti-SAG 1 mAb were significantly increased. Mice that received tachyzoties pretreated with both mAb and complement had longer survival times than those that received tachyzoites pretreated with mAb alone. Invasion of tachyzoites into fibroblasts and macrophages was significantly inhibited in the anti-GRA2, anti-GRA6 or anti-SAG 1 mAb pretreated group. Pretreatment with mAb and complement inhibited invasion of tachyzoites in both fibroblasts and macrophages. These results suggest that specific antibodies to dense-granule molecules may be useful for controlling infection with T. gondii.

• Parasites, Hosts and Diseases
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• 제35권2호
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• pp.79-86
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• 1997

톡소포자충의 숙주-기생충 상호관계 연구의 일환으로 마우스 비장에서 분리한 T 림프구. B 림프 구 및 과립구(대부분 호중구로 구성)에 톡소포자층의 tachyzoites를 감염시킨 후 감염된 림프구와 호중구의 미세형태 변화를 관찰하는 한편 각 세포의 충체 감염에 대한 감수성을 동위원소 흡수시 험법을 이용하여 정량화하였다. 충체는 병원성이 강한 RH 주를 샤용하였고 각 세포는 BALB/c와 CBA 마우스의 비장에서 분리하여 사용하였다. 감염 후 24시간에 관찰한 결과, T 림프구, B 림프 구 및 호중구는 마우스 주에 상관없이 세포질 내에 tachyzoites가 한 개, 두 개 또는 7-8개까지 관찰되었다. 감염된 T 림프구는 충체 주변에 형성죈 parasitophorous vacuole로 인해 핵이 한 쪽으로 밀리며. 미토콘드리아의 수가 증가하였다 감염된 B 림프구는 조내형질세망(RER)이 대조군에 비해 발달하지 않았으며 감염된 호중구는 과립의 수가 현저히 감소하였다 림프구와 호중구의 톡소포자충 감염에 대한 감수성을 3H-uracil 흡수량으로 정량화한 결과. 마우스 주에 따른 차이는 없었고 모든 종류의 세포 내에서 충체가 활발히 증식함이 확인되었다. 이상의 결과로 볼 때, BALB/c와 CBA 마우스의 비장 T 림프구, B 림프구 및 호중구는 모두 톡소포자충의 tachyzoites 감염에 대해 감수성이 높음을 알 수 있었고, 감염된 면역세포는 그 기능이 저하될 것으로 추측된다.

• Parasites, Hosts and Diseases
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• 제49권2호
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• pp.115-123
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• 2011

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 $1{\times}10^5$ tachyzoites) or day 11 PI (with $1{\times}10^6$ 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-Iymphocytes 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.

• Parasites, Hosts and Diseases
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• 제33권3호
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• pp.173-178
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• 1995

톡소포자충(RH strain) tachyzoltes를 감마선에 조사하여 인체 백혈병 세포인 HL-60 세포와 정상 마우스의 복강 대식세포에 감염시킨 다음 충체 증식 능력에 어떠한 변화가 오는지 $^3H-uracil$ 흡수시험을 통하여 알아보았다 감마선 조사량은 30, 70, 100및 300 Gy로 하였고 tachyzoite감염은 시켰으나 감마선을 조사하지 않은 비조사군(NI군)과 tachyzoites를 넣지 않고 숙주세포만을 배양한 비감염군을 각각 두었다. 3H-uracil 흡수시험 결과 12-24시간 후 대식세포의 NI 군은 2,190-4,787(countperminute. 이하 같음), HL-60세포의 NI 군은 2,967-8,254의 높은 흡수량을 보였으나. 감마선 조사군은 대식세포 감염시 381-703(100 Gy조사군) 및 218-408(300 Gy조사군), HL-60 세포 감염시 1,911-2,618(30 Gy), 1,253-1,384(70 Gy), 1,013-1,090(100 Gy) 및 483-588(300 Gy)의 흡수량을 각각 보였다. 충체 증식에 대한 억제율은 300 Gy 조사시 12-24시간에 대식세포의 경우 90-92%. HL-60 세포의 경우 80-94%이었다. 이상과 같이 톡소포자충 RH tachyzoites가 감마선 조사 후 세포내 분열증식 능력에 치명적인 영향을 받는다는 것을 uracil 흡수 시험법으로 확인하였다.