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Toxoplasma gondii is an obligate parasitic protozoon that transmits to animals and humans via ingested food. Cats that act as T. gondii's final hosts play a critical role in T. gondii transmission by shedding millions of oocysts. Timely diagnosis of infected cats is essential for preventing toxoplasmosis because oocysts are a putative T. gondii source in epidemiology. We developed a new visual LAMP assay targeting the B1 gene to analyze single oocysts in cat feces in this study. The amplification result could be visually estimated based on the color change. LAMP assay analytical sensitivity was 10 1 copies/µL for the B1 gene plasmid, which was tenfold better than the PCR reaction. There were no cross-reactions with other parasites. The LAMP assay can detect a single T. gondii oocyst in 200 mg of cat feces. The LAMP assay detected a single oocyst in 200 mg cat feces at a higher rate than the PCR assay (83.3% vs. 50.0%)., (© 2023. Springer Nature Limited.)

Background: Toxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-Associated Protein) systems are widely used for gene editing and pathogen detection. CRISPR-based diagnostics are molecular assays that have been developed to detect parasites with high sensitivity and specificity., Methods: This study aimed to establish a combined CRISPR/Cas12a and RPA rapid detection method for T. gondii by targeting the B1 gene and 529 bp repeat element (529 RE). The detection results could be visualized by the fluorescence or lateral flow strips (LFS). The sensitivity and specificity of the method were evaluated, and T. gondii-infected mouse blood was used for detection., Results: The results indicated that the established method for T. gondii detection was satisfactory, with a detection limit of 1.5 cp/μl for the two loci. Moreover, the B1 gene could detect 1 tachyzoite per reaction, and the 529 RE could detect 0.1 tachyzoite per reaction, consistently with the highly sensitive nested polymerase chain reaction (PCR) results. The method was suitable for strains, including RH, and did not cross-react with other protozoa DNA with similar habits. The T. gondii-infected mouse blood samples were all positive for T. gondii at 1, 3, and 5 days post infection (dpi)., Conclusions: This study established a rapid, sensitive, and time-saving DNA detection method for T. gondii that has the potential to be an alternative tool for T. gondii detection in the field., (© 2023. The Author(s).)

This study describes a simple method for the large-scale isolation of pure Toxoplasma gondii tachyzoites and bradyzoites. T. gondii tachyzoites were obtained from infected human foreskin fibroblasts (HFFs) and peritoneal exudates of mice, while tissue cysts containing bradyzoites were collected from chronically infected mice. Harvested cells and brain tissues were incubated in Hanks balanced salt solution (HBSS), containing 0.25% trypsin and 0.5% taurodeoxycholic acid (TDC) for 5 min. Subsequent washes in phosphate buffered saline (PBS) were conducted, and the cell viability of the preparations was good, as determined by flow cytometry and ability to reinfect HFF cells and propagate in mice. The purification procedure allowed for a rapid preparation of pure T. gondii tachyzoites and bradyzoites in sufficient quantity that can be used for downstream procedures. The advantage of the new method is that it is convenient and inexpensive., (© 2020 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect all nucleated cells through active invasion. Some non-canonical pathways for T. gondii infection of macrophages have recently been reported. We report a new mode of T. gondii invasion using a time-lapse imaging system, in which T. gondii tachyzoites are engulfed by a tube-like structure on peritoneal macrophage phagosomes and then escape from the phagosomes. Escaped parasites re-invade macrophages through intercellular junctions between their apical end and host cell membranes. We call this invasion pathway of T. gondii "pseudopod-assisted invasion" (PAI). The completion of this invasion process depends on parasitic motility and secretion of adhesins from parasitic micronemes. Our results provide new information about T. gondii infection and establish another platform for studying interactions between T. gondii and macrophages., (© American Society of Parasitologists 2020.)

Toxoplasma gondii (T. gondii) is a neurophilic and intracellular parasite that can affect plenty of vertebrate animals, including humans. Recent researches indicate that T. gondii infection is associated with neurodegenerative diseases such as Alzheimer's disease(AD). In addition, tau hyper-phosphorylation is a crucial event leading to the formation of nerve fiber tangles in AD. Despite the efforts to understand the interactions between T. gondii and AD, there are no clear results available so far. Here, we infected mice with the T. gondii of the Chinese 1 genotype Wh6 strain (TgCtwh6) for 60 days. Then we observed the formation of tissue cysts in the brain, the damage of neuron and the increased expression of phosphorylated tau (p-tau) in the hippocampal tissue of the mice. Similarly, we also found that p-tau, glycogen synthase kinase 3 beta (GSK3β), and phosphorylated GSK3β (p-GSK3β) were upregulated in vitro in TgCtwh6 challenged hippocampal neuron cell strain, HT22 cells. We noted a down-regulated expression of GSK3β,p-GSK3β, and p-tau in HT22 cells, which were pretreated with LiCl, an inhibitor of GSK3β. These data suggested that p-GSK3β may mediate tau phosphorylation after TgCtwh6 infection. Furthermore, TgCtwh6 infection also caused the increased expression of Bax and Caspase3, the decreased expression of Bcl-XL in HT22 cells, which had both early and late apoptosis. In all, our results indicated that TgCtwh6 infection not only led to phosphorylation of tau via activating GSK3β but also promoted hippocampal neuron apoptosis. Our research may partially reveal the mechanism with which TgCtwh6 induce neurofibrillary pathology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

As an indispensable molecular machine universal in all living organisms, the ribosome has been selected by evolution to be the natural target of many antibiotics and small-molecule inhibitors. High-resolution structures of pathogen ribosomes are crucial for understanding the general and unique aspects of translation control in disease-causing microbes. With cryo-electron microscopy technique, we have determined structures of the cytosolic ribosomes from two human parasites, Trichomonas vaginalis and Toxoplasma gondii, at resolution of 3.2-3.4 Å. Although the ribosomal proteins from both pathogens are typical members of eukaryotic families, with a co-evolution pattern between certain species-specific insertions/extensions and neighboring ribosomal RNA (rRNA) expansion segments, the sizes of their rRNAs are sharply different. Very interestingly, rRNAs of T. vaginalis are in size comparable to prokaryotic counterparts, with nearly all the eukaryote-specific rRNA expansion segments missing. These structures facilitate the dissection of evolution path for ribosomal proteins and RNAs, and may aid in design of novel translation inhibitors.

Toxoplasma gondii is an intra-cellular protozoan parasite that can infect almost all nucleated cells, eliciting host immune responses against infection. Host tissue damage is mainly caused by cellular lysis when T. gondii egresses from infected cells. However, the effects of cytokines released by host immune cells on egression of T. gondii remain elusive. This study aimed to investigate the role of tumor necrosis factor-alpha (TNF-α) on the egress of T. gondii from infected human foreskin fibroblast (HFF) cells and to elucidate the underlying mechanisms that regulate TNF-α-induced egress. Using flow cytometry to count tachyzoites of T. gondii released into cell culture medium, we found that egress of T. gondii from infected HFF cells could be induced by 10 ng/mL TNF-α in a time-dependent manner. Pre-treatment of infected HFF cells with BAPTA-AM to chelate intra-parasitic calcium could greatly inhibit TNF-α-induced egress. Similar results were obtained when using cytochalasin D to block parasite motility before the TNF-α-induced egress assay. In addition, blocking host apoptosis by Z-VAD-FMK could decrease TNF-α induced egress, while blocking necroptosis by necrostatin-1 has little impact on TNF-α-induced egress. The egressed tachyzoites displayed a normal growth rate and lost no virulence. Our results suggest that host cytokines could influence the cellular lytic processes of T. gondii, providing new insights into the relationship between host TNF-α and T. gondii pathogenesis., (© Y. Yao et al., published by EDP Sciences, 2017.)

Toxoplasma gondii converts from tachyzoites to bradyzoites after acute infection and thus survives the attack of the host immune responses. In this study, we observed the conversion of tachyzoites to bradyzoites in cell cultures using a transgenic T. gondii RH strain. The transgenic parasites continuously express yellow fluorescent protein (YFP) but only express red fluorescent protein (RFP) at the bradyzoite stage. Red fluorescent bradyzoite-containing cysts were found in transgenic parasite infected cells cultured with atmospheric CO2 supply, indicating the successful induction of the stage conversion. In cell culture with alkalic medium (pH 8.1) and atmospheric CO2 supply, only part of the YFP-expressing parasites in a cyst express RFP marker, suggesting the asynchronous development of T. gondii in vitro. This study provides a possibility for further studies of the gene expression profile during stage conversion and the genes involved.

Background: Egress is a vital step in the life cycle of Toxoplasma gondii which attracts attentions of many groups. Previous studies have shown that exogenous nitric oxide (NO) stimulates the early egress of T. gondii from infected peritoneal macrophages, a kind of immune cells. However, because Toxoplasma forms cysts in brain and muscle tissues, the development of autonomous immunity in non-immune cells is vital for limiting parasite burden and cyst formation. Therefore, we attempted to investigate whether exogenous NO could induce the early egress of T. gondii from infected non-immune cells., Methods: T. gondii tachyzoites were cultured in human foreskin fibroblast (HFF) cells and were then treated with NO released by sodium nitroferricyanide (III) dihydrate (SNP). The egressed parasites were analysed by flow cytometry., Results: The results showed that NO induced the early egress of parasites from HFF cells before completing their intracellular life cycles. We also found that the occurrence of egress was dependent on intracellular calcium (Ca(2+)) levels and the mobility of the parasite. Compared with freshly isolated tachyzoites, the developmental ability and virulence of egressed tachyzoites presented no difference., Conclusions: Taken together, our findings demonstrate a novel assay for the analysis of egress signalling mechanisms and an avenue of parasite clearance by hosts of T. gondii.