High levels of congenital transmission of Toxoplasma gondii in a commercial sheep flock

Our current understanding of congenital transmission of Toxoplasma gondii from ewe to lamb dictates that infection frequently results in abortion and the death of the developing foetus, that the birth of live infected lambs occurs rarely and that the cat is the predominant source of infection in ewes. Using direct polymerase chain reaction detection of T. gondii, we report high levels of congenital transmission occurring in a commercially managed sheep flock. We sampled foetal-derived placental tissue and tissues from aborted lambs and showed that congenital transmission was detected in these tissues from 61% of all pregnancies. Where pregnancies resulted in the death of one or more lambs, T. gondii was detected in the lamb tissue for all but one of 18 (94%) pregnancies. Of the successful pregnancies resulting in the birth of live lambs we were able to detect T. gondii in foetal-derived placental tissue from 37 of 70 (42%) pregnancies. These results show that congenital transmission is occurring in a high percentage of lambings including normal healthy lambings, at this farm, suggesting that this route of transmission from generation to generation may be much more significant than that reported previously. These results may have implications for sheep husbandry and future epidemiological studies of T. gondii.     

Toxoplasma gondii: an improved rat model of congenital infection

The objective of this study was to refine the rat model of congenital toxoplasmosis. In Fischer rats we found that visualization of spermatozoa in vaginal exudates and the detection of at least 6 g body weight increase between days 9 and 12 of pregnancy, allowed the diagnosis and timing of pregnancy with 60% specificity and 84% sensitivity. A dose of 10⁴Toxoplasma gondii bradyzoites or 10²T. gondii oocysts of the Prugniaud strain resulted in more than 50% of congenital infection of the rat litters. Transmission of T. gondii via lactation was not detected in rats inoculated with either bradyzoites or oocysts. Bioassays of 51 neonates born from mothers inoculated with bradyzoites (in tissue cysts) and 29 neonates from mothers inoculated with oocysts demonstrated that both liver and lungs can be used for the diagnosis of congenital transmission in this model.     

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.     

Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups.

Previous studies on Toxoplasma gondii population structure, based essentially on multilocus restriction fragment length polymorphism analysis or on multilocus enzyme electrophoresis, indicated that T. gondii comprises three clonal lineages. These studies showed a weak polymorphism of the markers (2-4 alleles by locus). In this study, we used eight microsatellite markers to type 84 independent isolates from humans and animals. Two microsatellite markers were present in the introns of two genes, one coding for beta-tubulin and the other for myosin A, and six were found in expressed sequence tags. With 3-16 alleles detected, these markers can be considered as the most discriminating multilocus single-copy markers available for typing T. gondii isolates. This high discriminatory power of microsatellites made it possible to detect mixed infections and epidemiologically related isolates. Evolutionary genetic analyses of diversity show that the T. gondii population structure consists of only two clonal lineages that can be equated to discrete typing units, but there is some evidence of occasional genetic exchange that could explain why one of these discrete typing units is less clearly individualised than the other.     

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.     

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。     

Toxoplasma gondii: partial cross-protection among several strains of the parasite against congenital transmission in a rat model

Rats were immunized with cysts of two Toxoplasma strains or with RH strain tachyzoites prior to pregnancy. The litters of the 13 rats that received homologous challenges with cysts during pregnancy, were all protected, whereas of 173 rats that received heterologous challenges with cysts or oocysts, only 21 protected their litters. 38.3 and 17% of rats immunized with the RH and with complete strains respectively, and 57% of control rats challenged with cysts, transmitted the infection congenitally. The percentages when similar groups were challenged with oocysts, were 33.3, 48.2, and 56.2%, respectively. Immunization with cysts did not completely protect against challenge with oocysts, even if the same strain was used. The divergence of these results from the complete protection against congenital toxoplasmosis observed in immune women and ewes, might be due to the use of excessive challenge doses in the model.     

Metabolic reconstruction identifies strain‐specific regulation of virulence in Toxoplasma gondii

Increasingly, metabolic potential is proving to be a critical determinant governing a pathogen's virulence as well as its capacity to expand its host range. To understand the potential contribution of metabolism to strain‐specific infectivity differences, we present a constraint‐based metabolic model of the opportunistic parasite, Toxoplasma gondii. Dominated by three clonal strains (Type I, II, and III demonstrating distinct virulence profiles), T. gondii exhibits a remarkably broad host range. Integrating functional genomic data, our model (which we term as iCS382) reveals that observed strain‐specific differences in growth rates are driven by altered capacities for energy production. We further predict strain‐specific differences in drug susceptibilities and validate one of these predictions in a drug‐based assay, with a Type I strain demonstrating resistance to inhibitors that are effective against a Type II strain. We propose that these observed differences reflect an evolutionary strategy that allows the parasite to extend its host range, as well as result in a subsequent partitioning into discrete strains that display altered virulence profiles across different hosts, different organs, and even cell types.     

Evaluation and applicability of a purification method coupled with nested PCR for the detection of Toxoplasma oocysts in water

AIMS: To describe the development, evaluation and applicability of a complete method for the detection of Toxoplasma gondii in water. METHODS AND RESULTS: The method incorporated concentration of water samples by Al(2)(SO(4))(3)-flocculation, purification by discontinuous sucrose gradients and detection of toxoplasmic DNA by 18S-rRNA nested PCR. Tap water replicates and natural water samples were seeded with defined numbers of Toxoplasma oocysts and processed for evaluation studies. When applied to environmental samples, the method gave highest detection sensitivities of 100 oocysts in river water and 10 oocysts in well- and sea water. The method was finally applied in 60 water samples of different quality and origin collected over a 14-month period. Toxoplasmic DNA was detected in four samples. CONCLUSIONS: The method offers an alternative towards improving current methods that can be used for the detection of Toxoplasma oocysts in environmental water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The method in its current form will be helpful for assessment of Toxoplasma contamination in water resources, particularly after outbreak events.     

Direct inhibition of cytochrome c-induced caspase activation in vitro by Toxoplasma gondii reveals novel mechanisms of interference with host cell apoptosis

The intracellular parasite Toxoplasma gondii is known to inhibit apoptosis of its host cell. The molecular mechanisms of this interference are, however, not yet completely understood. We show here that viable parasites prominently inhibited the activation of caspase 3/7 induced by cytochrome c, dATP and dithiothreitol in cytosolic extracts of human-derived Jurkat leukemic T cells. In contrast, granzyme B-induced caspase activity was only slightly diminished. De novo protein biosynthesis by T. gondii was dispensable for the inhibition of cytochrome c-induced caspase activation. Furthermore, a complete parasite lysate or, more importantly, molecules released by extracellular parasites mediated the interaction with the caspase cascade. The cell-free system applied here is thus a valuable tool to study the interaction of T. gondii and possibly other intracellular pathogens with host cell apoptosis.     

Identification of a human immunodominant B-cell epitope within the GRA1 antigen of Toxoplasma gondii by phage display of cDNA libraries.

Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection. With the aim of identifying the immunodominant epitopes of T. gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach. A library of cDNA fragments from T. gondii tachyzoites was displayed as fusion proteins to the amino-terminus of lambda bacteriophage capsid protein D. The lambdaD-tachyzoite library was then affinity-selected by using a panel of sera of pregnant women, all infected with the parasite. Some of the clones identified through this procedure matched the sequence of the dense granule GRA1 protein (p24), allowing us to identify its antigenic regions. In particular, the analysis of human antibody response against the recombinant GRA1 antigen fragments revealed the existence of an immunodominant epitope (epi-24 peptide).     

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．     

Regulation of Toxoplasma gondii multiplication in BeWo trophoblast cells: cross-regulation of nitric oxide production and polyamine biosynthesis

Materno-foetal transmission causes one of the most severe forms of infection with the protozoan parasite Toxoplasma gondii. Several studies have shown T. gondii in placental trophoblast cells, which form the barrier between maternal blood circulation and foetal tissue. Parasite multiplication in trophoblast cells is thus a critical step leading to infection of the foetus. Here, we show that multiplication of T. gondii tachyzoites was slow in BeWo trophoblast cells, compared with MRC-5 fibroblast cells. However, unlike MRC-5 cells, even combined stimulation with interferon-γ and tumor necrosis factor- did not reduce T. gondii replication in BeWo cells. This was associated with a lack of indoleamine-2,3-dioxygenase induction by these cytokines. Neither low availability of iron salts, nor an immunosuppressive action of cyclooxygenase-2 could be attributed to the low T. gondii multiplication rate in BeWo cells. However, treatment with the nitric oxide synthesis inhibitor N^G-methyl-l-arginine and addition of ornithine enhanced the proliferation rate of the intracellular pathogen. Despite detection of inducible nitric oxide synthase-II mRNA in BeWo cells, nitric oxide production could not be detected during cell culture. Thus, inhibition of arginase activity by nitric oxide synthesis may be partially responsible for the lower multiplication rate in BeWo cells.     

Non-canonical Maturation of Two Papain-family Proteases in Toxoplasma gondii

Proteases regulate key events during infection by the pervasive intracellular parasite Toxoplasma gondii. Understanding how parasite proteases mature from an inactive zymogen to an active enzyme is expected to inform new strategies for blocking their actions. Herein, we show that T. gondii cathepsin B protease (TgCPB) does not undergo self-maturation but instead requires the expression of a second papain-family cathepsin protease, TgCPL. Using recombinant enzymes we also show that TgCPL is capable of partially maturing TgCPB in vitro. Consistent with this interrelationship, antibodies with validated specificity detected TgCPB in the lysosome-like vacuolar compartment along with TgCPL. Our findings also establish that TgCPB does not localize to the rhoptries as previously reported. Accordingly, rhoptry morphology and rhoptry protein maturation are normal in TgCPB knock-out parasites. Finally, we show that although maturation of TgCPL is independent of TgCPB, it may involve an additional protease(s) in conjunction with self-maturation.     

Fitness of Toxoplasma gondii is not related to DHFR single-nucleotide polymorphism during congenital toxoplasmosis

Factors that regulate the pathogenesis of Toxoplasma gondii in humans are poorly understood. When acquired during pregnancy, toxoplasmosis can be disastrous, leading to fetal loss or conversely to subclinical disease. In congenitally infected infants, evolution is highly unpredictable. Genotype based virulence patterns have been described in mice, but in humans this classification does not correlate with the gravity of the disease. Mutations on DHFR-TS loci have recently been reported to confer T. gondii fitness cost. In this study, we investigated the relationship between the virulence of the parasite, as measured by clinical outcome in the fetus or newborn, fitness, as measured by parasitic load in amniotic fluid, and allelic polymorphism in DHFR. Six cases of severe congenital toxoplasmosis and 23 cases of mild congenital infections were included in the study. Quantitative PCR was performed to evaluate total T. gondii DNA load in amniotic fluid and detection of mutations was carried out with a LightCycler using hybridisation probes. Parasitic load was significantly higher in severe infections than in mild diseases. Among isolates from severe or non-severe cases of congenital toxoplasmosis, no polymorphism could be detected at loci 36, 83 or 245 of the DHFR gene. The virulent RH strain presented the same melting temperature as the non-virulent PRU strain for codons 36, 83 and 245. Only mutated clones, M2M3 and M2M4 with allelic replacement at these positions, displayed different profiles allowing a clear distinction between wild and mutant types. We concluded that the DHFR gene mutations we investigated do not regulate T. gondii fitness in humans.     

Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting.

, , , , , and 1992. Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting. International Journal for Parasitology 22: 1083–1088. Toxoplasma gondii trophozoites (RH strain) were cultured in embryonic fibroblasts in order to study the kinetics of production of excretory/secretory antigens, and the results were compared to the production of circulating antigens in an in vivo mouse model. By capture-ELISA, excretory/secretory antigens were first detected on the fourth day of culture whereas circulating antigens were first detected 1 day after infection. Similar concentrations of antigens were detected in both models as evidenced by comparable absorbance values. By immunoblotting, the excretory/secretory antigens were also detected later compared to circulating antigens (day 4 vs day 1). Seven major polypeptides were detected in both antigen preparations, six of them having the same molecular mass (110, 75,48, 30, 24 and 22 kDa).     

Analysis of the SAG5 locus reveals a distinct genomic organisation in virulent and avirulent strains of Toxoplasma gondii

We have recently characterised, in the virulent strain RH of Toxoplasma gondii, three glycosylphosphatidylinositol-anchored surface antigens related to SAG1 (p30) and encoded by highly homologous, tandemly arrayed genes named SAG5A, SAG5B and SAG5C. In the present study, we compared the genomic organisation of the SAG5 locus in strains belonging to the three major genotypes of T. gondii. Southern blot analysis using a SAG5-specific probe produced two related but distinct hybridisation patterns, one exclusive of genotype I virulent strains, the other shared by avirulent strains of either genotype II or genotype III. To understand the molecular bases of this intergenotypic heterogeneity, we cloned and sequenced the SAG5 locus in the genotype II strain Me49. We found that in this isolate the SAG5B gene is missing, with SAG5A and SAG5C laying contiguously. This genomic arrangement explains the hybridisation profiles observed for all the avirulent strains examined and indicates that the presence of SAG5B is a distinctive trait of genotype I. Furthermore, we identified two novel SAG1-related genes, SAG5D and SAG5E, mapping respectively 1.8 and 4.0 kb upstream of SAG5A. SAG5D is transcribed in tachyzoites and encodes a polypeptide of 362 amino acids sharing 50% identity with SAG5A-C, whereas SAG5E is a transcribed pseudogene. We also evaluated polymorphisms at the SAG5 locus by comparing the coding regions of SAG5A-E from strains representative of the three archetypal genotypes. In agreement with the strict allelic dimorphism of T. gondii, we identified two alleles for SAG5D, whereas SAG5A, SAG5C and SAG5E were found to be three distinct nucleotide variants. The higher intergenotypic polymorphism of SAG5A, SAG5C and SAG5E suggests that these genes underwent a more rapid genetic drift than the other members of the SAG1 family. Finally, we developed a new PCR–restriction fragment length polymorphism method based on the SAG5C gene that is able to discriminate between strains of genotype I, II and III by a single endonuclease digestion.     

Characterization of Toxoplasma gondii subtelomeric-like regions: identification of a long-range compositional bias that is also associated with gene-poor regions

Background

Chromosome ends are composed of telomeric repeats and subtelomeric regions, which are patchworks of genes interspersed with repeated elements. Although chromosome ends display similar arrangements in different species, their sequences are highly divergent. In addition, these regions display a particular nucleosomal composition and bind specific factors, therefore producing a special kind of heterochromatin. Using data from currently available draft genomes we have characterized these putative Telomeric Associated Sequences in Toxoplasma gondii.

Results

An all-vs-all pairwise comparison of T. gondii assembled chromosomes revealed the presence of conserved regions of ∼ 30 Kb located near the ends of 9 of the 14 chromosomes of the genome of the ME49 strain. Sequence similarity among these regions is ∼ 70%, and they are also highly conserved in the GT1 and VEG strains. However, they are unique to Toxoplasma with no detectable similarity in other Apicomplexan parasites. The internal structure of these sequences consists of 3 repetitive regions separated by high-complexity sequences without annotated genes, except for a gene from the Toxoplasma Specific Family. ChIP-qPCR experiments showed that nucleosomes associated to these sequences are enriched in histone H4 monomethylated at K20 (H4K20me1), and the histone variant H2A.X, suggesting that they are silenced sequences (heterochromatin). A detailed characterization of the base composition of these sequences, led us to identify a strong long-range compositional bias, which was similar to that observed in other genomic silenced fragments such as those containing centromeric sequences, and was negatively correlated to gene density.

Conclusions

We identified and characterized a region present in most Toxoplasma assembled chromosomes. Based on their location, sequence features, and nucleosomal markers we propose that these might be part of subtelomeric regions of T. gondii. The identified regions display a unique trinucleotide compositional bias, which is shared (despite the lack of any detectable sequence similarity) with other silenced sequences, such as those making up the chromosome centromeres. We also identified other genomic regions with this compositional bias (but no detectable sequence similarity) that might be functionally similar.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-21) contains supplementary material, which is available to authorized users.     

A Comparison of Cross Protection Between BCG,Hammondia hammondi,Besnoitia jellisoni and Toxoplasma gondii in Hamsters*

SYNOPSIS. The effect of pretreatment with BCG strain of Mycobacterium tuberculosis or Hammondia hammondi 21 days before challenge with lethal doses of Toxoplasma gondii and Besnoitia jellisoni was studied in hamsters. The results indicated that the intracardial administration of BCG provided no protection against either T. gondii or B. jellisoni. The hamsters immunized with H. hammondi survived challenge with 10⁴ lethal doses of T. gondii but only 1 lethal dose with B. jellisoni, indicating strong cross protection between H. hammondi and T. gondii and only a marginal one between H. hammondi and B. jellisoni.     

Conditional mutagenesis of a novel choline kinase demonstrates plasticity of phosphatidylcholine biogenesis and gene expression in Toxoplasma gondii

The obligate intracellular and promiscuous protozoan parasite Toxoplasma gondii needs an extensive membrane biogenesis that must be satisfied irrespective of its host-cell milieu. We show that the synthesis of the major lipid in T. gondii, phosphatidylcholine (PtdCho), is initiated by a novel choline kinase (TgCK). Full-length (～70-kDa) TgCK displayed a low affinity for choline (K(m) ～0.77 mM) and harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. Conditional mutagenesis of the TgCK gene in T. gondii attenuated the protein level by ～60%, which was abolished in the off state of the mutant (Δtgck(i)). Unexpectedly, the mutant was not impaired in its growth and exhibited a normal PtdCho biogenesis. The parasite compensated for the loss of full-length TgCK by two potential 53- and 44-kDa isoforms expressed through a cryptic promoter identified within exon 1. TgCK-Exon1 alone was sufficient in driving the expression of GFP in E. coli. The presence of a cryptic promoter correlated with the persistent enzyme activity, PtdCho synthesis, and susceptibility of T. gondii to a choline analog, dimethylethanolamine. Quite notably, the mutant displayed a regular growth in the off state despite a 35% decline in PtdCho content and lipid synthesis, suggesting a compositional flexibility in the membranes of the parasite. The observed plasticity of gene expression and membrane biogenesis can ensure a faithful replication and adaptation of T. gondii in disparate host or nutrient environments.