• Parasites, Hosts and Diseases
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• 제47권4호
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• pp.409-412
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• 2009

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.

• 대한수의학회지
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• 제41권1호
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• pp.89-98
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• 2001

This study was conducted to detect the toxoplasma specific-DNA in circulating blood and organs collected from slaughtered pigs at slaughtering house and experimentally infected pigs with Toxoplasma gondii tachyzoites by polymerase chain reaction(PCR), and also PCR was applied to diagnose for acute phase of swine toxoplasmosis as a newly developed diagnostic test. The sensitivity of oligonucleotide primer, T-1 & T-2, designed from toxoplasma B1 gene amplification method was compared with Tp parasite detection by mouse inoculation(MI). On the other hand, latex agglutination test(LAT) was conducted to detect the serum antibodies comparing with the detection of toxoplasma by PCR and MI. The results obtained were summarized as follows. PCR was able to determine at the lowest level of $10^0/ml$ T. gondii in blood samples which were blended with a serial diluted T gondii in vitro. On the other hand, $10^2/5g$ of T gondii could detect from a variety of tissues including lung, diaphragm, liver, heart, spleen and brain in vitro. The primer was proved to specifically determine T gondii in blood and tissues in vitro but it did not detect Neospora caninum used as a negative control. DNA of T. gondii was effectively extracted by freezing, thawing and grinding twice both tissues mixed with T gondii in vitro and in experimentally infected pig's tissues. PCR detected specific DNA in the blood of experimentally infected pigs at 108 hrs and 120 hrs post-infection, it was the same time that the pigs showed fever and parasitaemia. In case of tissue, specific DNA was, however, detected only lung from experimentally infected pigs. Even though the duration of acute phase was from 3 to 7 days post-infection, but the latex agglutination test (LAT) results appeared from 8 days post-infection. A comparison of sensitivity in determining T gondii in blood samples between PCR and MI, PCR positive rate ranged from 25 to 33.3%, but that of MI covered from 75 to 100%.

• Parasites, Hosts and Diseases
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• 제46권4호
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• pp.209-216
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• 2008

A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.

• 대한약침학회지
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• 제20권3호
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• pp.220-226
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• 2017

Objectives: The currently available agents for use against toxoplasmosis have serious limitations. Thus, the aim of the present study was to investigate the anti-Toxoplasma gondii (T. gondii) activities of methanol extracts of Feijoa sellowiana (F. sellowiana) (leaves and fruits), Quercus castaneifolia (Q. castaneifolia) (fruits), and Allium paradoxum (A. paradoxum) (leaves) in vitro and in vivo. Methods: Vero cells were treated with different concentrations (from 0 to $400{\mu}g/mL$) of the above extracts or with pyrimethamine at a dose of 50 mg/mL (positive control). Then, the viabilities of the T. gondii-infected cells were measured by using colorimetric MTT (3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide) assays. In addition, the survival rates of mice acutely infected with $2{\times}10^4$ RH strain tachyzoites of T. gondii were examined in vivo after intraperitoneal injection of the extracts at doses of 100 and 200 mg/kg/day for 5 days. Results: In the in vitro anti- T. gondii assay, the $IC_{50}$ values were 12.77, 180.2, 74.73, 213.2 and $163.8{\mu}g/mL$, and the selectivity indices were 6.05, 1.31, 0.35, 0.69 and 1.30 for the F. sellowiana (leaves and fruits), Q. castaneifolia, and A. paradoxum extracts and pyrimethamine, respectively. Moreover, the mice treated with F. sellowiana (leaves and fruits) achieved better results in terms of survival than the others (P < 0.05). Conclusion: The results of the current study indicate that methanol extract of F. sellowiana has significant anti-Toxoplasma activity. Further study should be conducted to investigate the potential bioactivity of this extract through bioactivity-guided fractionation.

• 대한수의학회지
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• 제39권6호
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• pp.1151-1160
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• 1999

This study was conducted to detect the toxoplasma-specific DNA in peripheral blood collected from cats experimentally infected with Toxoplasma gondii (RH strain) and from domiciled cats by B1 gene-base polymerise chain reaction(PCR). The sensitivity of oligonucleotide primer, T-1 & T-2, designed from toxoplasma B1 gene amplification method was compared with parasite detection by mouse inoculation(MI). And also, latex agglutination test(LAT) and indirect fluorescent antibody test(IFAT) were conducted to detect the fluctuation of serum antibodies compared with the detection of toxoplasma by PCR and MI. Toxoplasma B1 gene PCR was shown consistently high sensitivity and the results obtained by PCR agreed completely with those from MI. All blood samples collected before infection with T gondii gave negative results by PCR and MI. Also, toxoplasma Bl gene PCR was not cross reaction with Neospora caninum DNA and normal cat leucocyte as controls. The toxoplasma-specific DNA was detected by PCR in blood of 5 cats experimentally infected with T gondii 6 days after infection and the detection of this specific-DNA was long lasted in blood for 64 days after infection. The detection of toxoplasma-specific DNA by PCR could be identified as few as 10 tachyzoites and the isolation of T gondii by MI could be isolated as few as 1 tachyzoite from tenfold serial dilution of T gondii with normal cat blood, respectively. In healthy domiciled cats, the toxoplasma-specific DNA and the parasite were detected and isolated in blood from 3 of 56(5.3%) cats by both PCR and MI, respectively. In the results of antibody test from the total 56 heads of healthy domiciled cats, the positive rates are 15(26.7%) by LAT and 19(33.9%) by IFAT. These results suggest that PCR detection of toxoplasma can be applied as a sensitive and specific diagnostic and research tool.

• Parasites, Hosts and Diseases
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• 제25권2호
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• pp.188-194
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• 1987

돼지에 $1.5{\times}10^6$개의 Sarcocystis suicanis sporocyst와 10,000개의 Toxcplasma gondii oocyst(미국 Georgia 대학교 수의과대학 보존주)를 각각 경구감염 시킨후 9주간에 걸쳐 혈중 IgG 항체의 소장과 Sarcocystis와 Tohoplasma 사이의 혈청학적 교차반응성을 IFA 검사와 ELISA법을 이용하여 검사하였다. IFA검사와 ELISA를 위한 Sarcocystis의 전충체 항원과 수용성 항원은 감염돈의 확육을 인공소화액으로 소화시켜 준비하였다. Toxoplasma의 전충체 항원과 수용성 항원은 감염 mouse 복강에서 충체를 분리하여 사용하였다. Sarcocystis와 Togoplasma를 각각 인공감염시킨 돈혈청의 IgG 항체는 IFA검사와 ELISA에서 모두 감염 2주째에 처음으로 검출되었으며 Tcxoplasma에 대한 항체가는 감염 6주째에 최고치에 도달하였다가 그 이후에는 하강하였다. Sarcocystis에 대한 항체가는 감염 9주까지 서서히 상승하였다. Toxoplasma에 감염된 돈혈청은 Sarcocystis 항원에 대하여 IFA검사에서는 1 : 16까지 교차반응이 나타났으며 ELISA에서는 1 : 32까지 나타났다. 대조군의 돈혈청은 모두 1 : 4 이하이었다.

• Parasites, Hosts and Diseases
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• 제51권1호
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• pp.85-92
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• 2013

IL-23 and IL-12 are structurally similar and critical for the generation of efficient cellular immune responses. Toxoplasma gondii induces a strong cell-mediated immune response. However, little is known about IL-23 secretion profiles in T. gondii-infected immune cells in connection with IL-12. We compared the patterns of IL-23 and IL-12 production by THP-1 human monocytic cells in response to stimulation with live or heat-killed T. gondii tachyzoites, or with equivalent quantities of either T. gondii excretory/secretory proteins (ESP) or soluble tachyzoite antigen (STAg). IL-23 and IL-12 were significantly increased from 6 hr after stimulation with T. gondii antigens, and their secretions were increased with parasite dose-dependent manner. IL-23 concentrations were significantly higher than those of IL-12 at the same multiplicity of infection. IL-23 secretion induced by live parasites was significantly higher than that by heat-killed parasites, ESP, or STAg, whereas IL-12 secretion by live parasite was similar to those of ESP or STAg. However, the lowest levels of both cytokines were at stimulation with heat-killed parasites. These data indicate that IL-23 secretion patterns by stimulation with various kinds of T. gondii antigens at THP-1 monocytic cells are similar to those of IL-12, even though the levels of IL-23 induction were significantly higher than those of IL-12. The detailed kinetics induced by each T. gondii antigen were different from each other.

• Parasites, Hosts and Diseases
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• 제34권3호
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• pp.197-206
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• 1996

율무 수침엑스(Co-Ex)가 대식세포내 톡소포자충(Toxoplosmo gondii RH strain)의 번식에 억제 작용이 있다는 보고(Soh et al.., 1994)에 이어 본 연구는 Co-Ex내의 유효성분(effective component)을 추구하기 위하여 이미 탈지조작과 단백질 변성과정을 거친 Co-Ex를 Tris-buffer solution에 녹인 후 원심과정으로 그 상청(WC1) 그리고 WC1을 ammonium sulfate 처리 후 원심한 상청(WC2)과 침사(WC3)를 분리하였고 WC1은 다시 column chromatography에 의하여 WC4 WC5 WC6로 분획하여 시료로 사용하였으며. 대식세포 활성에 기여하는 것으로 이미 알려진 $IFN-{\gamma}$, LPS 등을 참고자료로 하였다. 각 시험자료들의 대식세포 활성화로 인한 반응질소 중간산물(RN1) 생산량. 대식세포의 톡소포자충 감염상 대식세포내 톡소포자충 번식상 등을 비교 조사하였다 10% FCS 첨가 DMEM 배지에 배양하였고 이에 각종 시료(BRM)를 실험목적에 따라 배지에서 24시간 작용시킨 뒤 RNI를 조사하였고 이어 톡소포자충 $1\times10^6$을 30분 감염시킨 뒤 새 배지에 옮겨 48시간 배양한 뒤 Pl, Fl 등을 조사하였다. NO 생산은 각례 대조(시료 비투여)에 비하여 높았고 $IFN-{\gamma}$ 첨가는 생산량(${\mu}M/l$)을 더욱 증가시켰다(예 Control 7.5 Control + $IFN-{\gamma}$ 14.2 WC2 26.2 WC2 + $IFN-{\gamma}$ 57.8). 포자충감염율(%)은 대조에 비하여 낮았고 $IFN-{\gamma}$ 첨가시에는 더욱 낮아졌는데 $IFN-{\gamma}$ 첨가예에서의 감염율은 대조 38.0인데 비하여 WC2 19.2. WC1 + WC2 + WC3 7.2로 나타났다. 세포당감염율도 같은 경향으로 INF-$\gamma$ 첨가시험에서 대조 6.0(감염 대식세포 100개 중의 톡소포자충 평균수) WC2 1.7, WCI + WC2 + WC3 1.4 등으로 나타 났다. 결론적으로 율무엑스 수침엑스 중 WC2, WC1 등은 대식세포를 활성화하나 $IFN-{\gamma}$ 첨가는 더욱 그 활성화를 높이며 그 중 WC2가 활성화에 주역을 하는 것으로 사료되는 바이다.

• Parasites, Hosts and Diseases
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• 제33권2호
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• pp.107-116
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• 1995

국내 마우스에서 분리된 작은와포자충(Cwptospoyidinpown)을 마우스에서 증식시킨 다음 오오시스트만을 순수 분리하여 BAIB/c 마우스에 면역시켰다. 면역된 마우스의 비장에서 림프구를 분리하여 PEG 1500을 융합촉진제로 사용하여 Sp2/0 myeloma cell과 세포융합을 실시하였으며, 융합된 세포중 작은와포자충에 특이항체를 생산하는 hybridomacell을 선발하였다 한계희석법으로 제작된 2주의 단세포군항체는 IgG2b class(lE7.2)와 IgM class(C6)에 속했으며. SDS-PAGE와 Westernblotting한 결과 (IE7.2)는 원충의 단백항원중 36 kDa과 반응하였고, C6는 67 kDa 및 70 kDa과 반응하였다 생산된 단세포군항체를 간접 형광항체법으로 작은와포자충과 반응시킨 결과 오오시스트 외막체 특이적으로 반응하였던 반면, Tomplasma gondii의 tachyzoite, Eimeria zuernii E. bouis, E. conodensis의 오오시스트와는 반응을 나타내지 않았다. 단세포군항체 C6을 이용한 간접 형광항체법은 분변의 이물들의 데조염색을 위해 Inns blue를 사용하였으며, 관찰소견으로는 $3-5{\mu\textrm{m}}$의 등근 오오시스트가 9.l은 형광을 띄고 있었고 그외의 주변이물들은 대조염색에 의하여 검붉게 염색되었다 또한 그의 진단율은 현재 일반적으로 사용되고 있는 수입진단킷트(Merifluor, Meridian diagnostic Ins.)와 거의 일치하고 있었다. 이상의 결과를 종합해보면 이번에 생산된 단세포군항체 들은 작은와포자충에 특이적으로 반응하고 있었으며, 이를 이용한 형광항체 진단법은 작은와포자충을 진단하는데 유용하게 사용될 수 있을 것으로 판단된다.

• Parasites, Hosts and Diseases
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• 제55권5호
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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.