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.     

Kinetics of the growth of Toxoplasma gondii (RH strain) in mice.

A method is described for obtaining maximum yield of Toxoplasma tachyzoites from the peritoneal cavity of infected mice. Mice injected with 10² parasites contain more Toxoplasma in this site at the time of death than mice injected with larger numbers. The host does not mount a detectable humoral response to the parasite.     

Attenuation of the virulent RH strain of Toxoplasma gondii by passages in mice immunized with Toxoplasma lysate antigens

The virulent RH strain of Toxoplasma gondii was attenuated after a few passages or just one long passage in mice immunized twice with a four-week interval between immunizations with an emulsion of Toxoplasma lysate antigens and complete Freund's adjuvant. Three avirulent strains, RH-cyst III, IV and VIII were established from the RH strain. The RH-cyst III strain was effective for vaccination against challenge with the original, virulent RH strain. The attenuation of T. gondii is an expression of the innate attributes of this parasite necessary to maintain its parasitic life cycle in nature.     

Infection and immunity with the RH strain of Toxoplasma gondii in rats and mice.

Infection and immunity to toxoplasmosis induced by the RH strain of Toxoplasma gondii was compared in Sprague-Dawley (SD) and Wistar rats and in outbred Swiss Webster mice. All rats injected with up to 1,000,000 RH-strain tachyzoites remained clinically normal, whereas mice injected with only 1 live tachyzoite died of acute toxoplasmosis. Rats could be infected with 1 tachyzoite of the RH strain as shown by antibody development and by bioassay in mice. However, after 8 days, RH-strain organisms were recovered only inconsistently from SD and Wistar rat brains. Contrary to a report of sterile immunity to T. gondii infection in rats after immunization with live RH tachyzoites, we found infection immunity after challenge with the VEG strain. Toxoplasma gondii tissue cysts of the VEG strain could be recovered from most SD and Wistar rats, first injected with live RH-strain tachyzoites and then challenged with oocysts of the VEG strain. Our RH strain, and probably many others, passed for 50+ yr as tachyzoites has lost not only the capacity to form oocysts, but also shows a marked reduction or absence of tissue cyst (bradyzoites) formation.     

Mode of Action of Ponazuril Against Toxoplasma gondii Tachyzoites in Cell Culture

ABSTRACT. Toxoplasma gondii is an important apicomplexan parasite of humans and other warm-blooded animals. Ponazuril is a triazine anticoccidial recently approved for use in horses in the United States. We investigated the inode of action of ponazuril against developing RH strain T. gondii tachyzoites in African green monkey kidney cells. Host cells were infected with  2.0 × 10⁵  tachyzoites and treated with 5 μg/ml ponazuril. Cultures were fixed and examined by transmission electron microscopy 3 days after treatment. Ponazuril interfered with normal parasite division. This led to the presence of multinucleate schizonts stages. Up to six tachyzoites were observed partially budded from the surface of these schizonts. Large vacuoles developed in these schizonts and they eventually degenerated.     

Detection of antibodies to the 97 kDa component of Toxoplasma gondii in samples of human serum

This study was carried out to investigate the immune response against 97 kDa (p97) molecular marker of Toxoplasma gondii that has been characterized as a cytosolic protein and a component of the excreted-secreted antigens from this parasite. A total of 60 serum samples from patients were analyzed by enzime-linked immunosorbent assay and Western blot for toxoplasmosis. These samples were organized in three groups, based on clinical symptoms and results of serological tests. Group I: 20 samples reactive to IgG and IgM (acute phase); group II: 20 non-reactive samples (control group); and group III: 20 samples reactive only to IgG (chronic phase). Western blot was performed with total antigenic extracts or with excreted and secreted antigen from T. gondii to identify the fraction correspondent to p97. It was observed that this cytosolic component from T. gondii stimulates the immunologic system to produce both IgM and IgG antibodies in the beginning of the acute infection and IgG throughout the chronic stage of the asymptomatic toxoplasmosis.     

The prevalence of antibodies against Toxoplasma gondii in some Ontario mammals

A survey of serum samples from mammals trapped in Central Ontario showed that many contained antibodies to Toxoplasma gondii. The prevalence of infection as reflected by positive reactions in the Sabin-Feldman Dye Test appeared to be related to the type of diet of each species examined, and specifically, to the proportion of rodents in the diet. Of the fox blood samples tested, 84% were positive. The percentage of positive samples diminished through, coyote, mink, bear, fisher skunk, raccoon, marten and rabbit. Blood samples from squirrel, deer, hare and groundhog were negative.     

Monoclonal antibodies against nucleoside triphosphate hydrolase-II can reduce the replication of Toxoplasma gondii

Nucleoside triphosphate hydrolase (NTPase) is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii, which has a wide specificity toward NTP. In the present study, two monoclonal antibodies (mAbs, MNT1 and MNT2) against recombinant T. gondii NTPase-II (rTgNTPase-II) were developed. Western blot analysis displayed that these two mAbs can recognize specifically rTgNTPase-II as well as a 63 kDa molecule in tachyzoites soluble antigens that corresponded to native NTPase-II. T. gondii tachyzoites pretreated with two mAbs were observed under Confocal Laser Microscope and a specific reaction was displayed on tachyzoites after indirect fluorescence antibody test (IFAT). When COS-7 cells were co-cultured with tachyzoites pretreated with two mAbs, the number of intracellular parasites per infected cell was significantly decreased compared with the control. Furthermore, incubation of T. gondii tachyzoites with two mAbs can inhibit NTPase activity in the presence of dithiothreitol, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the mAbs. The passive immunization test indicated that the transferred mAbs can significantly prolong the survival time of challenge infected mice. Taken together, we concluded that the mAbs against NTPase-II can reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.     

弓形虫RH株SAG1基因序列体外扩增及生物信息学分析

根据sAG1基因序列,自行设计一对寡核苷酸引物,利用PCR技术从弓形虫RH株基因组DNA中成功扩增出编码SAG1抗原的基因片段,扩增出的基因片段大小与预期长度（1006bp）相符,结果经测序验证,并利用生物信息学方法对SAG1蛋白理化性质、结构和功能进行了预测．     

Toxoplasma gondii RH Ankara: production of evolving tachyzoites using a novel cell culture method

Nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) are present in the food vacuole (FV) of Plasmodium falciparum trophozoites. The product of PFL1555w, a putative cytochrome b₅, localizes in the FV membrane, similar to what was previously observed for the product of PF13_0353, a putative cytochrome b₅ reductase. These two gene products may contribute to NO generation by denitrification chemistry from nitrate and/or nitrite present in the erythrocyte cytosol. The possible coordination of NO to heme species present in the food vacuole was probed by resonance Raman spectroscopy. The spectroscopic data revealed that in situ generated NO interacts with heme inside the intact FVs to form ferrous heme nitrosyl complexes that influence intra-vacuolar heme solubility. The formation of heme nitrosyl complexes within the FV is a previously unrecognized factor that could affect the equilibrium between soluble and crystallized heme within the FV in vivo.     

Serum antibodies against Toxoplasma gondii and Neospora caninum in southeast Queensland dugongs

The dugong (Dugong dugon) is an herbivorous marine mammal that inhabits tropical inshore waters and thus may be vulnerable to pollutants and terrestrial pathogens as a result of coastal runoff. In this study, serum samples collected from live, wild dugongs (n = 114) in an embayment located on the urbanized southeast Queensland coast of Australia during 2008–2014, were measured for IgG antibody levels specific to Toxoplasma gondii and Neospora caninum. An ELISA used to measure T. gondii tachyzoite antibodies indicated a non-Gaussian distribution of antibody level, with five dugongs identified as high outliers. Mean levels of antibodies specific for T. gondii in dugongs sampled in 2014 were significantly higher than in 2010 (p = .006) and 2011 (p = .009) with an elevation in mean antibody levels after a major 2011 flood event relative to antibody levels prior to the flood (p < .0001). A competitive ELISA to detect N. caninum antibody indicated a normal distribution of antibody with no high outliers. Mean antibody level for N. caninum was highest in 2012 and declined significantly in 2014 (p = .004). This is the first survey of antibodies directed against T. gondii and N. caninum in dugongs and suggests future health monitoring of this species.     

A recombinant pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma gondii RH strain and pseudorabies in BALB/c mice

The major immunodominant surface antigen 1 (TgSAG1) of invasive tachyzoites is a vaccine candidate antigen for Toxoplasma gondii. In this study, we developed a recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV/SAG1) based on the PRV vaccine strain Bartha K-61 by homologous recombination, in which partial PK and gG genes were deleted. The growth assay of rPRV/SAG1 showed that the recombinant virus can replicate in vitro as efficiently as PRV Bartha K-61, demonstrating that insertion of the TgSAG1 gene in the PK and gG locus of PRV does not affect the replication of PRV. All mice vaccinated with rPRV/SAG1 developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong increase of the splenocyte proliferative response, and significant levels of IFN-gamma and IL-2 production. And the immunization of mice with rPRV/SAG1 elicited strong cytotoxic T lymphocyte (CTL) responses in vitro. These results demonstrate that rPRV/SAG1 could induce significant humoral and cellular Th1 immune responses. Moreover, rPVR/SAG1 immunization induced partial protection (60%) against a lethal challenge with the highly virulent T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. These results suggest that expression of protective antigens of T. gondii in PRV Bartha K-61 is a novel approach towards the development of a vaccine against both animal toxoplasmosis and pseudorabies.     

Prevalence of Toxoplasma gondii antibodies in dingoes

Serum samples from 62 dingoes (Canis familiaris dingo) trapped in five areas of southeastern New South Wales, Australia were tested for antibodies to Toxoplasma gondii. Six (10%) of the dingoes had direct agglutination test titers for T. gondii of greater than or equal to 1:64, and four of these animals had T. gondii-specific IgM, suggesting recent exposure.     

A piezoelectric immunoagglutination assay for Toxoplasma gondii antibodies using gold nanoparticles

The serologic detection of anti-Toxoplasma gondii immunoglobulins plays a key role in the clinical diagnosis of Toxoplasmosis. In this paper, a simple, rapid and highly sensitive agglutination-based piezoelectric immunoassay has been firstly developed for directly detecting anti-T. gondii immunoglobulins in infected rabbit serum (IRS) and infected rabbit blood (IRB). The proposed technique is based on that the specific agglutination of antigen-coated gold nanoparticles, averaging 10nm in diameter, in the presence of the corresponding antibody causes a frequency change that is monitored by a piezoelectric device. In contrast to the commonly used piezoelectric assays, it possesses an attractive advantage in that the immobilization of antibody or antigen on the crystal is unnecessary. Use of a newly prepared sensing probe which was modified by a plasma-polymerized film (PPF) of n-butyl amine and further by a heparin layer resulted in a response-enhanced immunoagglutination and a high compatibility of the probe with biological samples. An appropriate reagent consisting of 1% normal rabbit serum (NRS) and 0.1% bovine serum albumin (BSA) for diluting the analytes were verified in counteracting the background interference of assay. Moreover, an optimization of assay medium composition with the addition of poly(ethylene glycol) (PEG) serving as immunoagglutination rate and sensitivity enhancer was investigated in detail. It is found that the developed immunoagglutination assay system is sensitive to dilution ratio of anti-T. gondii antibody as low as 1:5500. Analytical results of several specimens obtained using the developed technique are in satisfactory agreement with those given by the ELISA method, implying a promising alternative approach for detecting anti-T. gondii antibodies in the clinical diagnosis.     

Divalent Cation and ATP Dependent Motility of Toxoplasma gondii Tachyzoites After Mild Treatment with Trypsin

ABSTRACT. Large percentages of Toxoplasma gondii tachyzoites could be induced to display two types of movement associated with active invasive behavior by exposing them for 1 min to 0.002% trypsin in phosphate-buffered saline (PBS). The motile activity, consisting of clockwise rotation around the posterior end (about 20 revolutions per min) and twirling-gliding over a poly-L-lysine substrate (1.2 ± 0.2 μm/s standard deviation), was observed and recorded by video-enhanced contrast microscopy. The number of active tachyzoites reached a maximum 1 min after trypsinization; the motile response of the population lasted for about 5 min. Activation was prevented by soybean trypsin-inhibitor, and could not be induced again in previously treated specimens. Electron-microscopy of trypsinized tachyzoites fixed in the presence of ruthenium-red revealed discrete discontinuities of the plasma membrane, which sealed within 90 min after washing with PBS. Treated tachyzoites were able to invade cultured epithelial cells with a higher relative infectivity than that of untreated parasites. Perfusion of trypsinized tachyzoites with 1 mM of either CaCl₂ or MgCl₂ and 1 mM ATP increased the number of activated parasites to over 60%; on the other hand, all induced motility was inhibited or blocked by agents that chelate divalent cations. The present preparation, which provided the first serial illustrations of T. gondii movements induced by a defined chemical stimulus, may offer a useful experimental model for the study of motility in this parasite.     

Ca2+-Dependence of Conoid Extrusion in Toxoplasma gondii Tachyzoites

The role of Ca²⁺ in conoid extrusion was investigated in isolated Toxoplasma gondii tachyzoites by treatment with Ca²⁺-ionophores, Ca²⁺-chelating agents and an inhibitor of the Ca²⁺-ATPase at the endoplasmic reticulum. The results were evaluated by light phase-contrast microscopy and electron microscopy. lonomycin (0.5-1 μM) caused an immediate and sustained extrusion of the conoid in up to 80% of the tachyzoites, depending on the concentrations of ionophore and Ca²⁺ in the medium. However, over 50% of the tachyzoites extruded the conoid when treated with ionomycin in Ca²⁺-free saline complemented with EGTA. The effect of ionomycin was reversible and could be induced a second time in about half of the responsive population. Similar results were obtained with A23187. Conoid extrusion induced by ionomycin in Ca²⁺-free medium was almost completely abolished when the tachyzoites were previously loaded with a permeable compound known to chelate intracellular Ca²⁺ (BAPTA/AM; 25μM). On the other hand, exposure of tachyzoites to the Ca²⁺-ATPase inhibitor thapsigargin (0.5-1μM) produced significant extrusion of the conoid. Tachyzoites loaded with BAPTA/AM as well as those treated with ionomycin, i.e. with conoids paralyzed in opposite positions, had a diminished capacity to invade cultured epithelial cells. A substantial reduction in the response to stimulation by ionomycin was found also in parasites treated with cytochalasin-D, a drug that depolymerizes actin-filaments. The results suggest that Ca²⁺-release from internal stores may act as a key signal to activate a mechanism of conoid extrusion probably mediated, at least in part, by actin-filaments.     

Prevalence of Toxoplasma gondii antibodies in domestic cats from rural Ohio

Antibodies to Toxoplasma gondii were determined in serum samples from 275 domestic cats from a mobile spay and neuter clinic from 8 counties in Ohio. The modified agglutination test incorporating whole formalinized tachyzoites and mercaptoethanol was used to determine antibodies. Antibodies to T. gondii were found in 133 (48%) out of 275 cats: in titers of 1:25 in 24, 1:50 in 37, and 1:500 or more in 72. The highest prevalence (62% of 78) was in outdoor cats. The prevalence of T. gondii antibodies in 48% of cats suggests wide-spread contamination of the rural environment with oocysts.     

Prevalence of antibodies to Toxoplasma gondii in dogs from northeastern Portugal

Prevalence of antibodies to Toxoplasma gondii was investigated in 673 domestic dogs from northeastern Portugal, using the modified agglutination test (MAT) with 1 : 20 as cutoff for seropositivity; antibodies were found in 256 dogs (38.0%). Differences between seroprevalence levels in males (36.7%) and females (41.8%) and between pure-breed (42.1%) and mixed-breed dogs (35.2%) were not statistically significant. Multiple logistic regression analysis identified age above 12 mo (odds ratio [OR] = 4.0), chance of eating birds or small mammals (OR = 4.0), housing exclusively outdoors (OR = 1.5), home-cooked meals (OR = 3.0), and eating raw meat or viscera (OR = 7.7) as risk factors for the canine T. gondii infection. Some control measures are suggested based on these findings.     

Cytochemical Localization of Plasma Membrane Enzyme Markers During Interiorization of Tachyzoites of Toxoplasma gondii by Macrophages

The enzyme activity of Mg-ATPase, Na⁺-K⁺-ATPase, 5'-nucleotidase and NAD(P)H-oxidase was cytochemically detected at the ultrastructural level in mouse peritoneal macrophages infected with untreated and with specific antibody-coated Toxoplasma gondii tachyzoites. The Mg⁺⁺-ATPase and 5'-nucleotidase were distributed throughout the macrophages'plasma membrane but were not observed in the membrane lining endocytic vacuoles containing ingested parasites; however, Na⁺-K⁺-ATPase activity was detected in the macrophages'plasma membrane as well as in the parasitophorous vacuoles that contained untreated or specific antibody-coated parasites. Reaction product, indicative of NAD(P)H-oxidase, was detected in the parasitophorous vacuoles that contained only-specific antibody-coated parasites.     

Toxoplasma gondii: cross-immunity against the enteric cycle

Eight of nine cats inoculated with strain ME-49 and challenged with three different strains of Toxoplasma were immune to oocyst shedding, as ascertained with bioassays of their feces. In a second experiment, only toxoplasma asexual stages were seen in H&E stained gut sections of cats treated with suppressive doses of sulfamerazine and pyrimethamine starting 2 days after oral inoculation with cysts of the strain ME-49 and killed 6 days later. In a third experiment, four cats were similarly inoculated and treated for 20 days. Six weeks later, the cats received an oral homologous challenge with cysts, and none shed toxoplasma oocysts. An acceptable level of cross-protection was achieved with strain ME-49, and therefore, it can be used as a candidate strain from which antigens could be tested for enteric protection.