Construction of Toxoplasma gondii bradyzoite expressing the green fluorescent protein

The bradyzoite stage of Toxoplasma gondii is a key step in the parasite life cycle. For a better understanding of this stage, a sensitive system to detect the tissue cysts would be required. In this study, we generated the T. gondii cyst-forming strain PLK expressing green fluorescent protein (GFP) under control of the dense granule protein 1 promoter, which works at both the tachyzoite and the bradyzoite stages. The bradyzoites with GFP fluorescence within both small and large cysts were detectable in the brain of mice infected with the recombinant PLK. Indeed, the bradyzoites expressing GFP had infectivity to mice. This study shows that transfection of the cyst-forming strain with GFP gene under control of the GRA1 promoter could be a useful approach for the study of the bradyzoite stage of T. gondii.     

Development of an in vitro model of Toxoplasma gondii cyst formation

Abstract Toxoplasma gondii tissue cyst reactivation is a major pathogenic mechanism in ocular toxoplasmosis, disease associated with AIDS and organ transplantation. The mechanisms associated with cyst formation and reactivation have not been elucidated. The complexity of studying these issues in animal models has led to the development of in vitro tissue culture strategies for cyst formation. In the present study we have adopted the human embryonic lung fibroblast (HEL) as the host cell and have compared the cyst forming abilities of eight clinical isolates. We describe by transmission electron microscopy and quantitative light microscopy the development of cysts in vitro. The numbers of in vitro cysts increased with time for all isolates. Cyst cultures were stabilised by manipulation of the free parasite load, an observation not previously recorded. Thus, in this paper we describe a viable model for the analysis of the mechanisms of Toxoplasma cyst development.     

Experimental tissue cyst induced Toxoplasma gondii infections in dogs

Tissue cyst induced Toxoplasma gondii infections were examined in 2 beagle dogs orally inoculated with tissue cysts. Neither dog developed clinical signs of toxoplasmosis. Both dogs developed low antibody titers to T. gondii. The MAT and IFAT were superior to the LAT and IHT tests for detecting antibodies to T. gondii.     

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．     

Feline Toxoplasmosis from Acutely Infected Mice and the Development of Toxoplasma Cysts*

SYNOPSIS. The development of Toxoplasma cysts was studied in mice inoculated with tachyzoites by several routes. After 1–30 days of infection, murine tissues were examined microscopically, and portions or whole carcasses were fed to mice and cats. The feces of the cats were examined for oocyst shedding. Cyst-like structures containing distinct PAS-positive granules were first seen after 3 days of infection with tachyzoites, and became numerous by 6 days. Argyrophilic walls were first seen after 6 days, and became numerous by 16 days of infection with tachyzoites. Prepatent periods to oocyst shedding (PPO) were either “short” (3–10 days) or “long” (19–48 days). The “short” PPO was found only in cats that had ingested mice infected for 3 days or longer, and was related to the development of PAS-positive granules in T. gondii, and to high, 60–100%, oral infectivity rates for cats. The “long” PPO followed the ingestion of mice infected for only 1–2 days, and was related to tachyzoites without distinct PAS-positive granules and low, 32% or less, infectivity for cats. The “long” PPO followed also the ingestion of oocysts and the parenteral inoculation of tachyzoites, bradyzoites, or sporozoites. Using the “short” PPO as a criterion for detecting cysts in tissues, it was shown that (a) numerous cysts developed in mice 5 days after inoculation with tachyzoites, 7–9 days after inoculation with cysts, and 9–10 days after inoculation with oocysts, and (b) cysts developed faster and more frequently in the brain and muscle than in lungs, liver, spleen, and kidneys of mice inoculated with tachyzoites.     

Loss of Stages after Continuous Passage of Toxoplasma gondii and Besnoitia jellisoni*

SYNOPSIS. Toxoplasma gondii, passed from mouse to mouse in the tachyzoite stage for 30–35 generations, developed cysts, which, when fed to cats, failed to produce oocysts. Besnoitia jellisoni, passed similarly for 20 generations, lost the capacity to form cysts. These phenomena are explained by a loss of genomes or gene products during the rapid passage selecting for tachyzoites.     

A Comparative Electron Microscopic Study of the Morphology of Toxoplasma gondii by Freeze-Etch Replication and Thin Sectioning Technic

SYNOPSIS. Freeze-etch preparations of Toxoplasma gondii reveal details of structure and organelles in 3-dimensional relationships. The subpellicular microtubules and their relationship to the polar ring, the tripartite pellicle, the pellicle constituents, and the spatial relationship of the rhoptries to the conoid and conoid canal are clearly demarcated.     

弓形虫感染实验动物模型的特点及建模方案的选择

弓形虫感染对人类生活和畜牧业发展构成严重威胁。弓形虫感染实验动物模型是进行弓形虫学相关研究的基础条件之一。在实际研究工作中,根据不同的实验目的、选取不同的实验动物和以不同的实验方法所建立的实验动物模型,呈现出复杂和多变的特点。这一方面可以满足不同实验的需要,但同时也在实验结果的评价上导致一定程度的不足。根据弓形虫感染实验动物模型的不同特点,针对特定的实验目的,选择适合方法建立适合的实验动物模型,是进行相关弓形虫学研究的有效基础和前提。     

弓形虫ZS2和RH株基因差异表达的研究

从RH和ZS2株弓形虫抽提纯化mRNA,经反转录后获得的双链cDNA分别作为driver和tester,采用抑制消减杂交技术(suppression substraction hybridization,SSH)技术扩增ZS2株中差异表达的基因。电泳图谱显示消减的cDNA与未消减的cDNA PCR扩增产物存在明显的差异,说明ZS2和RH株弓形虫转录水平存在着差异。     

弓形虫（Toxoplasma gondii）的PCR检测方法在笼养猕猴群中的应用

目的建立快速、灵敏、特异及检测结果易判断的PCR方法,并应用于大规模猕猴种群的弓形虫常规检测中。同时比较巢式PCR和单一PCR的一致性。方法根据弓形虫保守基因p30（SAG1）设计了内、外两对进行巢式PCR扩增以及B1基因设计一对引物进行单一PCR扩增,将DNA样本进行10倍倍比稀释,以检测巢式PCR反应的灵敏度;并对医学生物学研究所自繁猕猴共150只进行了弓形虫检测。结果巢式PCR检测法检测限度可达10^-3ng/uL,而且方法特异。两种PCR法检测结果基本一致,其中巢式PCR检测阳性率（10％）稍高于单一PCR检测阳性率（8．67％）。结论巢式PCR和一次PCR方法都可应用于猕猴弓形虫的常规检测中,并提示巢式PCR比单一PCR更敏感、检出率更高。     

弓形虫上海株的棒状体蛋白ROP2的克隆表达及纯化

根据已发表基因序列(GenBank登录号为Z36906)设计引物,以弓形虫(Toxoplasma gondii)上海本地株的基因组DNA为模板,扩增编码ROP2(rhpotry protein2)蛋白的基因片段,定向克隆至表达质粒pET32a(+),重组质粒经限制性酶切鉴定后测序,结果表明插入片段长度为1044bp,与GenBank上登录的序列相比,同源性为96%-100%,其中与弓形虫RH株的rop2基因同源性为100%。重组原核表达质粒pET32a-rop2转化至大肠杆菌BL21(DE3),经诱导可表达分子量约60.9kD的融合蛋白,能被感染弓形虫RH株的绵羊阳性血清识别。     

Evaluation of a rapid ELISA technique for detection of circulating antigens of Toxoplasma gondii

To evaluate a modified rapid ELISA method for detecting CAg during Toxoplasma gondii infection, we analyzed the specificity and sensitivity of the ELISA method by using experimental Toxoplasma infection in rabbits and also tested this method in human samples including 5428 serum, 548 cerebrospinal fluid and two breast milk samples. We prepared PcAb, and used it for rapid one-step sandwich ELISA testing in which an incubation time in the regular ELISA procedure was omitted. This method detected CAg at the concentration of 31.2 ng/mL, and no cross-reaction was found with antigens of protozoa (Cryptosporidium parvum, Plasmodium falciparum), trematode (Schistosoma japonicum, Paragonimus sp.) and nematode (Brugia malayi, Ancylostoma duodenale, Ascaris lumbricoides and Trichinella spiralis). CAg was detected in rabbit serum 3 days after infection, and optical density values reached a peak 9-13 days after infection, then declined gradually. Among human serum samples, the positive rate of CAg was 2.11% in cerebral paralysis patients, whereas it was 0.22% or 0.71% in patients without neurological symptoms or in uncomplicated pregnant women. The difference among these three groups was statistically significant (P < 0.05). The positive rate of cerebrospinal fluid samples from cerebral paralysis patients was 10.58%. There is a statistically significant difference between the positive rates of meat-packing workers and blood donors (P < 0.01). In the retrospective analysis, CAg was detected in accordance with the onset of clinical symptoms, suggesting that CAg could reflect the clinical course in humans. Together with these results, CAg detected in the modified rapid sandwich ELISA could be a sensitive marker for acute and active infection of T. gondii.     

Toxoplasma gondii lacks the enzymes required for de novo arginine biosynthesis and arginine starvation triggers cyst formation

Two separate carbamoyl phosphate synthetase activities are required for the de novo synthesis of pyrimidines and arginine in most eukaryotes. Toxoplasma gondii is novel in possessing a single carbamoyl phosphate synthetase II gene that corresponds to a glutamine-dependent form required for pyrimidine biosynthesis. We therefore examined arginine acquisition in T. gondii to determine whether the single carbamoyl phosphate synthetase II activity could provide both pyrimidine and arginine biosynthesis. We found that arginine deprivation efficiently blocks the replication of intracellular T. gondii, yet has little effect on long-term parasite viability. Addition of citrulline, but not ornithine, rescues the growth defect observed in the absence of exogenous arginine. This rescue with citrulline is ablated when parasites are cultured in a human citrullinemia fibroblast cell line that is deficient in argininosuccinate synthetase activity. These results reveal the absence of genes and activities of the arginine biosynthetic pathway and demonstrate that T. gondii is an arginine auxotroph. Arginine starvation was also found to efficiently trigger differentiation of replicative tachyzoites into bradyzoites contained within stable cyst-like structures. These same parasites expressing bradyzoite antigens can be efficiently switched back to rapidly proliferating tachyzoites several weeks after arginine starvation. We hypothesise that the absence of gene activities that are essential for the biosynthesis of arginine from carbamoyl phosphate confers a selective advantage by increasing bradyzoite switching during the host response to T. gondii infection. These findings are consistent with a model of host-parasite evolution that allowed host control of bradyzoite induction by trading off virulence for increased transmission.     

Toxoplasma gondii myosin F,an essential motor for centrosomes positioning and apicoplast inheritance

Members of the Apicomplexa phylum possess an organelle surrounded by four membranes, originating from the secondary endosymbiosis of a red alga. This so‐called apicoplast hosts essential metabolic pathways. We report here that apicoplast inheritance is an actin‐based process. Concordantly, parasites depleted in either profilin or actin depolymerizing factor, or parasites overexpressing the FH2 domain of formin 2, result in loss of the apicoplast. The class XXII myosin F (MyoF) is conserved across the phylum and localizes in the vicinity of the Toxoplasma gondii apicoplast during division. Conditional knockdown of TgMyoF severely affects apicoplast turnover, leading to parasite death. This recapitulates the phenotype observed upon perturbation of actin dynamics that led to the accumulation of the apicoplast and secretory organelles in enlarged residual bodies. To further dissect the mode of action of this motor, we conditionally stabilized the tail of MyoF, which forms an inactive heterodimer with endogenous TgMyoF. This dominant negative mutant reveals a central role of this motor in the positioning of the two centrosomes prior to daughter cell formation and in apicoplast segregation.     

Quantitative immunolocalization of four immunodominant antigens of Toxoplasma gondii

Ultrastructural localization of four immunodominant antigens of Toxoplasma gondii was investigated quantitatively on thin sections and replicas by an immunogold technique using four monoclonal antibodies (Mab). On immunoblot Mab IV47, GII9, II38 and IE10 identified proteins of 28, 30, 45 and 66-70 kDa, respectively. Use of digital image analyzer and a semi-automatic procedure developed by us, the patterns of label distribution were compared in three cell structures: cell surface, submembrane area and rhoptries. On the whole cell surface, protein P28 and P30 were 2.5 and 4 times more abundant than P66-70 respectively. The protein P28 was essentially concentrated in the submembrane area with a labeling of 195.4 +/- 46.7 gold particles/microns 2 that follows a decreasing gradient from this area to the cell centre. In the rhoptries, all four antigens were detected, P45 and P66-70 being major with a labeling of 97.1 +/- 31.1 gold particles/microns 2 and 155.1 +/- 39.3 gold particles/microns 2 respectively. The results support the hypothesis that rhoptries are the essential site for antigen storage.     

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways^1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca²⁺, a signal that is also critical for host cell invasion^6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress⁹. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches^10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress¹³. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology^11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine^16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 °C), but fail to proliferate at the restrictive temperature (40 °C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype¹³. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites¹¹. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell¹⁹. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed²⁰. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.     

Specific Labeling of Intracellular Toxoplasma gondii with Uracil*

SYNOPSIS Radioactive uracil was not significantly incorporated into the nucleic acids of human fibroblast cells. Infection of these cells with Toxoplasma gondii resulted in an exponential increase in the rate of uracil incorporation that paralleled the exponential growth of the parasite. One day after infection the rate of uracil incorporation was increased 100-fold. It was established by autoradiography that all of the [³H] uracil was incorporated into the intracellular parasites. A possible explanation for this difference in ability to use uracil is our observation that the specific activity of uridine phosphorylase was 100-fold greater in partially purified parasites than in the host cell.     

Differential detection of Hammondia hammondi from Toxoplasma gondii using polymerase chain reaction

Hammondia hammondi and Toxoplasma gondii are two related coccidian parasites, with cats as definitive hosts and warm-blooded animals as intermediate hosts. It is difficult to differentiate them by morphological and serological parameters. In the present study, primers were designed to specifically amplify the ITS-1 region of H. hammondi to differentiate it from T. gondii. Attempts were made to detect the presence of H. hammondi DNA in the tissues of mice infected with H. hammondi alone, as well as from mixed infections with T. gondii, using the newly designed primers. The de novo primers effectively amplified the H. hammondi-specific target fragment from all samples containing H. hammondi, including those with concomitant T. gondii infection. Further, the primers did not amplify any fragment from the related parasites like T. gondii, Neospora caninum and Hammondia heydorni. The new primers provide simple and efficient means to differentially diagnose H. hammondi from T. gondii even in samples containing both parasites, thus obviating the need for other labourious techniques like mouse bioassay and in vitro cultivation.     

Amplification of the secretory IgA response to Toxoplasma gondii using cholera toxin

Our study demonstrates that cholera toxin (CT) markedly enhances the intestinal anti-T. gondii antibody response following oral immunisation of mice with a T. gondii sonicate (TSo) and CT. The antibodies induced were mostly IgA and secretory IgA but a small quantity of IgG was also produced. In contrast, no intestinal anti-T. gondii IgM antibodies were detected. Anti-CT IgA antibodies were also present in intestinal secretions but in much lower quantities than the T. gondii-specific IgA. No anti-CT IgG nor IgM antibodies were detected. Western blot analysis showed that CT induced not only an increase of the intensity of the intestinal IgA antibody response to the 30-kDa band but also induced intestinal IgA antibodies against other major T. gondii proteins (p22, and the 28-kDa antigen) as recognised by specific monoclonal antibodies. The amplification of the anti-T. gondii secretory IgA response by means of an appropriate adjuvant may be one major step leading towards an orally induced immune protection against toxoplasmosis.     

Effect of Immunization of Cats with Isospora felis and BCG on Immunity to Reexcretion of Toxoplasma gondii Oocysts*

SYNOPSIS. The effect of pretreatment with Isospora felis and bacillus Calmette-Guérin (BCG) on the reexcretion of Toxoplasma gondii occysts was studied in 16 coccidia-free cats. The following conclusions were drawn: (A) Chronically T. gondii-infected cats reexcreted T. gondii oocysts after superinfection with I. felis, and this reexcretion was prevented in cats infected with I. felis before T. gondii infection. (B) Administration of BCG before Toxoplasma infection had no apparent effect on the outcome of the infection.