• Parasites, Hosts and Diseases
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• v.50 no.3
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• pp.199-205
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• 2012

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.

• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.623-632
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• 1993

This study was conducted to develop a sensitized latex-antigen for serodiagnosis of toxoplasmosis in animals. Tachyzoites of T gondii(RH-strain) harvested from mouse peritoneal cavity were purified through the filtraton of polycarbonate membrane(pore size, $3.0{{\mu}m}$, Costar Co.) and disrupted by ultrasonicator. The tachyzoite suspension was ultracentrifuged for 30 min at $60,000{\times}g(4{^{\circ}C})$ and the supernatant was used as a water-lysate antigen. Polystyrene latex particles of $0.8{{\mu}m}$ in diameter(Sigma) were used for the preparation of sensitized latex-antigen suspension. The several parameters including the preparation conditions, incubation buffer. serum dilution buffer and stability of agglutination reactions were evaluated and the results obtained were summarized as follows : 1. The antigen consisting of a water-lysate of T gondii tachyzoites was adsorbed onto polystyrene latex particles of $0.8{{\mu}m}$ in diameter by adding a latex suspension to an equal volume of diluted antigen solution and by incubating the mixture at $37{^{\circ}C}$ under different conditions. 2. The optimum incubation buffer used for the antigen sensitization was 0.1M Tris-HCl buffer(pH 8.0). 3. The optimum serum dilution buffer used for the latex agglutination test was 0.1M Tris-HCl-NaCl buffer(pH 7.4) containing 300 mM NaCl. But 0.1M Tris-HCl-NaCl buffer(pH 7.4) containing 300-600 mM NaCl, 0.5% BSA and 0.01% Tween-20 improved the agglutination pattems and cleared the background of microplate well without the effects on L.A titer. 4. The time required for antigen sensitization was 40 and 60 min in incubation buffer(pH 8.0) at $37{^{\circ}C}$. But the optimun time for antigen sensitization was min at $37{^{\circ}C}$. 5. The optimun quantity of antigen absorbed on latex particles for proper agglutination was the range of 20 to $32{\mu}g$ of latex particles. 6. The optimun concentration of the latex-antigen suspension for the proper agglutination reaction was determined as 0.2%(w/v). 7. The specificity, rapidity and simplicity of the latex-particle agglutination test suggested that it might be adaptable to large scale serum screening.

• The Journal of the Korean Society for Microbiology
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• v.20 no.1
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• pp.103-107
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• 1985

Female ICR mice were vaccinated against Toxoplasma gondii(RH strain), infected 2 weeks later and after recovery mated to normal ICR males. Control matings were with normal ICR females. The progeny of the above matings were weaned at 1 week, vaccinated at 1, 2, 3, 4 or 5 weeks of age and infected 2 weeks later with lethal Toxoplasma tachyzoites. As assessed by survival, the effectiveness of vaccination among offspring of vaccinated-recovered mothers was greatly impaired than that of control mice, with respective of age : where mice did survive, recovery was greatly delayed relative to the controls. The protective effect of vaccination among infants born to control mothers was also blocked by maternal specific antibodies, by administration of high-titered specific antibodies or by transfer of nylon wool adherent immune-spleen cells, but was augmented by transfer of nylon wool passed immune-spleen cells. These results indicate that this impairment of vaccination may be due to the transmission of maternal specific antibodies to the offspring which acts to suppress both priming by the vaccine and the generation of parasite-specific helper T cells.

• 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.52 no.5
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• pp.493-499
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• 2014

The determination of the accurate immune status of pregnant women is crucial in order to prevent congenital toxoplasmosis. Equivocal results with conventional serological techniques are not uncommon when IgG titers are close to the cut-off value of the test, so that a confirmatory technique is needed. For this purpose, we developed a homemade immunoblot (IB) using soluble extract of Toxoplasma gondii tachyzoites and assessed it by testing 154 positive, 100 negative, and 123 equivocal sera obtained from pregnant women. In order to select the more valuable bands in terms of sensitivity and specificity, we used the Youden Index (YI). The highest YIs were those given by the 32, 36, 98, 21, and 33 bands. The simultaneous presence on the same blot of at least 3 bands showed a much higher YI (0.964) and was adapted as the positivity criterion. The analysis of results showed that our homemade IB correlated well with the commercial LDBIO Toxo II $IgG^{(R)}$ kit recently recommended as a confirmatory test (96.7% of concordance).

• Korean Journal of Veterinary Research
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• v.37 no.4
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• pp.817-823
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• 1997

Suckling piglets and weaned pigs showed anorexia, dehydration, severe abdominal breathing, emaciation and paresis from Oct. 1993. to Nov. 1993. Five 2-week-old piglets were submitted for diagnosis in Kangwon National University. At necropsy, the pin-point well demarcated yellowish white foci were scattered on the surface of the lung, heart, liver, spleen and kidney. Histologically, multifocal areas of necrosis with mononuclear cells infiltration were found in the lung, heart, liver, lymph node, spleen, kidney and small intestine. These lesions tended to be associated with blood vessels. Variable round to ovoid tachyzoites were located at the periphery of the lesions. The organisms were demonstrated as Toxoplasma gondii by immunohistochemical staining method. Ultrastructurally, this parasite was surrounded with parasitophorous vacuole in alveolar macrophage. The parasite was crescent-shaped and $6{\sim}8{\times}1{\sim}2{\mu}m$ in size. It was enclosed by an thick outer membrane and an underlying thin inner membrane. Several club-shaped paired organelles and conoids lay in the cytoplasm at the anterior. Numerous round body and one to several mitochondria were presented in the cytoplasm. Based on the gross findings, histopathology, immunohistochemical and electron microscopic findings, this case was diagnosed as toxoplasmosis in piglets.

• Parasites, Hosts and Diseases
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• v.41 no.3
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• pp.165-169
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• 2003

This study describes the characterization of 80 kDa pretense showing gelationlytic property among three pretenses in the excretory/secretory proteins (ESP) from Toxoplasma gondii. The pretense activity was detected in the ESP but not in the somatic extract of RH tachyzoites. This pretense was active only in the presence of calcium ion but not other divalent cationic ions such as $Cu^{2+},{\;}Zn^{2+},{\;}Mg^{2+},{\;}and{\;}$Mn^{2+}$, implying that $Ca^{2+}$ is critical factor for the activation of the protease. The 80 kDa pretense was optimally active at pH 7.5. Its gelatinolytic activity was maximal at $37^{\circ}C$, and significant level of enzyme activity of the pretense remained after heat treatment at $56^{\circ}C$ for 30 min or $100^{\circ}C$ for 10 min, This thermostable enzyme was strongly inhibited by metal chelators, i.e., EDTA, EGTA, and 11 10-phenanthroline. Thus, the 80 kDa pretense in the ESP secreted by T. gondii was classified as a calcium dependent neutral metalloprotease.

• Parasites, Hosts and Diseases
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• v.41 no.2
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• pp.89-96
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• 2003

Among the panel of monoclonal antibodies (mAb) against Toxoplasma gondii, mAb of Tg621 (Tg621) clone blotted 38 kDa protein which localized in the cytoplasm of tachyzoites by immunofluorescence microscopy The protein was not released into the parasitophorous vacuole during or after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg621. The full length cDNA sequence was completed with 5’-RACE as 1,592 bp, which contained open reading frame of 942 bp. The deduced amino acid sequence of Tg621 consisted of a polypeptide of 313 amino acids, with significant homology to ribosomal P proteins (RPP) of other organisms especially high to those of apicomplexan species. The expressed and purified TgRPP was assayed in western blot with the sera of toxoplasmosis patients and normal sera, which resulted in the 74.0% of positive reactions in toxoplasmosis patients whereas 8.3% in normal group. Therefore, the antibody formation against TgRPP in toxoplasmosis patients was regarded as specific for T. gondii infection and suggested a potential autoantibody.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.607-612
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• 1997

A case of neosporosis is described in a 6-month-old aborted bovine fetus. Grossly, numerous, well-demarcated, 2-3mm, yellowish white foci were scattered in the heart and skeletal muscle. Histopathologic changes were confined to the brain, heart, and skeletal muscle. Multifocal areas of necrotizing encephalitis were observed in the cerebrum as well as cerebellum. Severe multifocal to coalescing nonsuppurative inflammation was observed in the myocardium and skeletal muscle. The tachyzoites of Neospora caninum were demonstrated in the brain, myocardium and skeletal muscle with immunohistochemical method using Neospora caninum-specific antibody. Based on the histopathology and immunohistochemistry, this case was diagnosed as neosporosis in aborted Korean cattle. This is believed to be the first reported case of bovine neosporosis in Korea.

• Korean Journal of Veterinary Research
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• v.40 no.1
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• pp.138-144
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• 2000

This study was carried out to investigate the pathogenicity of Korea N caninum isolates, KBA-1 and KBA-2 on SCID mouse following 3 different routes of infection. NC-1 was served as reference isolate. The pathogenecity was evaluated by progression of clinical signs, histopathology and immunohistochemistry. Pathogenicity of KBA-2 appears to be stronger than that of KBA-1 but weaker than that of NC-1. Progress of clinical signs and lesion distribution and pattern of each isolates were similar when the isolates were infected either subcatareously or intraperitoneally. However, oral inoculation of tachyzoites failed to induce the infection.