Evaluation of protective immunity induced by a DNA vaccine encoding SAG2 and SRS2 against Toxoplasma gondii infection in mice.

Toxoplasma gondii is an important protozoan pathogen, which can cause severe diseases in the newborns and immunocompromised individuals. Developing an effective vaccine against Toxoplasma infection is a critically important global health priority. Immunofluorescence staining analysis revealed that TgSAG2 and TgSRS2 are membrane associated and displayed on the surface of the parasite. Immunizations with pBud-SAG2, pBud-SRS2 and pBud-SAG2-SRS2 DNA vaccines significantly increased the production of specific IgG antibodies. Immunization with pBud-SAG2-SRS2 elicited cellular immune response with higher concentrations of IFN-γ and IL-4 compared to the control group. Antigen-specific lymphocyte proliferations in the pBud-SRS2 and pBud-SAG2-SRS2 groups were significantly higher compared to that in the control group. Furthermore, 30 % of mice immunized with pBud-SAG2-SRS2 survived after the challenge infection with virulent T. gondii RH tachyzoites. This study revealed that immunization with pBud-SAG2-SRS2 induced potent immune responses, and has the potential as a promising vaccine candidate for the control of T. gondii infection.

Anti-Toxoplasma gondii effect of tylosin in vitro and in vivo.

BACKGROUND: Toxoplasma gondii is an important protozoan pathogen with medical and veterinary importance worldwide. Drugs currently used for treatment of toxoplasmosis are less effective and sometimes cause serious side effects. There is an urgent need for the development of more effective drugs with relatively low toxicity. METHODS: The effect of tylosin on the viability of host cells was measured using CCK8 assays. To assess the inhibition of tylosin on T. gondii proliferation, a real-time PCR targeting the B1 gene was developed for T. gondii detection and quantification. Total RNA was extracted from parasites treated with tylosin and then subjected to transcriptome analysis by RNA sequencing (RNA-seq). Finally, murine infection models of toxoplasmosis were used to evaluate the protective efficacy of tylosin against T. gondii virulent RH strain or avirulent ME49 strain. RESULTS: We found that tylosin displayed low host toxicity, and its 50% inhibitory concentration was 175.3 µM. Tylsoin also inhibited intracellular T. gondii tachyzoite proliferation, with a 50% effective concentration of 9.759 µM. Transcriptome analysis showed that tylosin remarkably perturbed the gene expression of T. gondii, and genes involved in "ribosome biogenesis (GO:0042254)" and "ribosome (GO:0005840)" were significantly dys-regulated. In a murine model, tylosin treatment alone (100 mg/kg, i.p.) or in combination with sulfadiazine sodium (200 mg/kg, i.g.) significantly prolonged the survival time and raised the survival rate of animals infected with T. gondii virulent RH or avirulent ME49 strain. Meanwhile, treatment with tylosin significantly decreased the parasite burdens in multiple organs and decreased the spleen index of mice with acute toxoplasmosis. CONCLUSIONS: Our findings suggest that tylosin exhibited potency against T. gondii both in vitro and in vivo, which offers promise for treatment of human toxoplasmosis.

Expression profiles of host miRNAs and circRNAs and ceRNA network during Toxoplasma gondii lytic cycle.

Toxoplasma gondii is an opportunistic protozoan parasite that is highly prevalent in the human population and can lead to adverse health consequences in immunocompromised patients and pregnant women. Noncoding RNAs, such as microRNAs (miRNAs) and circular RNAs (circRNAs), play important regulatory roles in the pathogenesis of many infections. However, the differentially expressed (DE) miRNAs and circRNAs implicated in the host cell response during the lytic cycle of T. gondii are unknown. In this study, we profiled the expression of miRNAs and circRNAs in human foreskin fibroblasts (HFFs) at different time points after T. gondii infection using RNA sequencing (RNA-seq). We identified a total of 7, 7, 27, 45, 70, 148, 203, and 217 DEmiRNAs and 276, 355, 782, 1863, 1738, 6336, 1229, and 1680 DEcircRNAs at 1.5, 3, 6, 9, 12, 24, 36, and 48 h post infection (hpi), respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed that the DE transcripts were enriched in immune response, apoptosis, signal transduction, and metabolism-related pathways. These findings provide new insight into the involvement of miRNAs and circRNAs in the host response to T. gondii infection.

Comprehensive analysis of mRNA-lncRNA co-expression profiles in mouse brain during infection with Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular protozoan parasite which seriously threatens the health of domestic animals and humans. Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts greater than 200 nucleotides, which are widely involved in transcriptional and epigenetic regulations. However, little is known about the roles of host lncRNAs in the response to T. gondii infections. In this study, using Illumina sequencing technology, we analyzed the expression profiles of mRNAs and lncRNAs in BALB/c mouse brain following infection by T. gondii PRU strain (type II genotype) cysts. The identified differentially expressed (DE) RNAs were subjected to bioinformatics analysis. A total of 2,090 annotated lncRNAs along with 3,577 novel lncRNAs were identified. In the acutely infected mouse brain, a total of 330 mRNAs and 19 lncRNAs were dys-regulated, whereas 136 DE mRNAs and 9 DE lncRNAs were identified in chronically infected mouse brain. GO analysis revealed that these DE mRNAs identified at acute infection stage were involved in immune response, whereas DE mRNAs found at chronic infection stage were mostly enriched in response to protozoan. KEGG analysis showed that DE mRNAs were significantly enriched in disease related pathways. In addition, the putative mRNA-lncRNA co-expression network was constructed, and several hub regulatory RNAs were identified based on the transcriptome data. This study firstly characterized the co-expression profile of mRNAs and lncRNAs in mouse brain infected with T. gondii and provided a framework for further studies of the roles of lncRNAs in host neuropathology during toxoplasmosis progression.

A combined miRNA-piRNA signature in the serum and urine of rabbits infected with Toxoplasma gondii oocysts.

BACKGROUND: Increasing evidence has shown that non-coding RNA (ncRNA) molecules play fundamental roles in cells, and many are stable in body fluids as circulating RNAs. Study on these ncRNAs will provide insights into toxoplasmosis pathophysiology and/or help reveal diagnostic biomarkers. METHODS: We performed a high-throughput RNA-Seq study to comprehensively profile the microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) in rabbit serum and urine after infection with Toxoplasma gondii oocysts during the whole infection process. RESULTS: Total RNA extracted from serum and urine samples of acutely infected [8 days post-infection (DPI)], chronically infected (70 DPI) and uninfected rabbits were subjected to genome-wide small RNA sequencing. We identified 2089 miRNAs and 2224 novel piRNAs from the rabbit sera associated with T. gondii infection. Meanwhile, a total of 518 miRNAs and 4182 novel piRNAs were identified in the rabbit urine associated with T. gondii infection. Of these identified small ncRNAs, 1178 and 1317 serum miRNAs and 311 and 294 urine miRNAs were identified as differentially expressed (DE) miRNAs in the acute and chronic stages of infections, respectively. A total of 1748 and 1814 serum piRNAs and 597 and 708 urine piRNAs were found in the acute and chronic infection stages, respectively. Of these dysregulated ncRNAs, a total of 88 common DE miRNAs and 120 DE novel piRNAs were found in both serum and urine samples of infected rabbits. CONCLUSIONS: These findings provide valuable data for revealing the physiology of herbivore toxoplasmosis caused by oocyst infection. Circulating ncRNAs identified in this study are potential novel diagnostic biomarkers for the detection/diagnosis of toxoplasmosis in herbivorous animals.

Investigation of urine metabolome of BALB/c mouse infected with an avirulent strain of Toxoplasma gondii.

BACKGROUND: The protozoan parasite Toxoplasma gondii is a major concern for human and animal health. Although the metabolic understanding of toxoplasmosis has increased in recent years, the analysis of metabolic alterations through noninvasive methodologies in biofluids remains limited. METHODS: Here, we applied liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics and multivariate statistical analysis to analyze BALB/c mouse urine collected from acutely infected, chronically infected and control subjects. RESULTS: In total, we identified 2065 and 1409 metabolites in the positive electrospray ionization (ESI +) mode and ESI - mode, respectively. Metabolomic patterns generated from principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) score plots clearly separated T. gondii-infected from uninfected urine samples. Metabolites with altered levels in urine from T. gondii-infected mice revealed changes in pathways related to amino acid metabolism, fatty acid metabolism, and nicotinate and nicotinamide metabolism. CONCLUSIONS: This is the first study to our knowledge on urine metabolic profiling of BALB/c mouse with T. gondii infection. The urine metabolome of infected mouse is distinctive and has value in the understanding of Toxoplasmosis pathogenesis and improvement of treatment.

Temporal transcriptomic changes in long non-coding RNAs and messenger RNAs involved in the host immune and metabolic response during Toxoplasma gondii lytic cycle.

BACKGROUND: Long non-coding RNAs (lncRNAs) are important regulators of various biological and pathological processes, in particular the inflammatory response by modulating the transcriptional control of inflammatory genes. However, the role of lncRNAs in regulating the immune and inflammatory responses during infection with the protozoan parasite Toxoplasma gondii remains largely unknown. METHODS: We performed a longitudinal RNA sequencing analysis of human foreskin fibroblast (HFF) cells infected by T. gondii to identify differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs), and dysregulated pathways over the course of T. gondii lytic cycle. The transcriptome data were validated by qRT-PCR. RESULTS: RNA sequencing revealed significant transcriptional changes in the infected HFFs. A total of 697, 1234, 1499, 873, 1466, 561, 676 and 716 differentially expressed lncRNAs (DElncRNAs), and 636, 1266, 1843, 2303, 3022, 1757, 3088 and 2531 differentially expressed mRNAs (DEmRNAs) were identified at 1.5, 3, 6, 9, 12, 24, 36 and 48 h post-infection, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DElncRNAs and DEmRNAs revealed that T. gondii infection altered the expression of genes involved in the regulation of host immune response (e.g., cytokine-cytokine receptor interaction), receptor signaling (e.g., NOD-like receptor signaling pathway), disease (e.g., Alzheimer's disease), and metabolism (e.g., fatty acid degradation). CONCLUSIONS: These results provide novel information for further research on the role of lncRNAs in immune regulation of T. gondii infection.

Quantitative Peptidomics of Mouse Brain After Infection With Cyst-Forming Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite capable of establishing persistent infection within the host brain and inducing severe neuropathology. Peptides are important native molecules responsible for a wide range of biological functions within the central nervous system. However, peptidome profiling in host brain during T. gondii infection has never been investigated. Using a label-free peptidomics approach (LC-MS/MS), we identified a total of 2,735 endogenous peptides from acutely infected, chronically infected and control brain samples following T. gondii infection. Quantitative analysis revealed 478 and 344 significantly differentially expressed peptides (DEPs) in the acute and chronic infection stages, respectively. Functional analysis of DEPs by Gene Ontology suggested these DEPs mainly originated from cell part and took part in cellular process. We also identified three novel neuropeptides derived from the precursor protein cholecystokinin. These results demonstrated the usefulness of quantitative peptidomics in determining bioactive peptides and elucidating their functions in the regulation of behavior modification during T. gondii infection.

Urine proteomics for profiling of mouse toxoplasmosis using liquid chromatography tandem mass spectrometry analysis.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis. Urine is an easily obtained clinical sample that has been widely applied for diagnostic purposes. However, changes in the urinary proteome during T. gondii infection have never been investigated. METHODS: Twenty four-hour urine samples were obtained from BALB/c mice with acute infection [11 days post infection (DPI)], mice with chronic infection (35 DPI) and healthy controls, and were analyzed using a label-free liquid chromatography tandem mass spectrometry analysis. RESULTS: We identified a total of 13,414 peptides on 1802 proteins, of which 169 and 47 proteins were significantly differentially expressed at acute and chronic infection phases, respectively. Clustering analysis revealed obvious differences in proteome profiles among all groups. Gene ontology analysis showed that a large number of differentially expressed proteins (DEPs) detected in acute infection were associated with biological binding activity and single-organism processes. KEGG pathway enrichment analysis showed that the majority of these DEPs were involved in disease-related and metabolic pathways. CONCLUSIONS: Our findings revealed global reprogramming of the urine proteome following T. gondii infection, and data obtained in this study will enhance our understanding of the host responses to T. gondii infection and lead to the identification of new diagnostic biomarkers.

Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry-Based Metabolomics Reveals Metabolic Alterations in the Mouse Cerebellum During Toxoplasma gondii Infection.

Toxoplasma gondii is a protozoan parasite with a remarkable neurotropism. We recently showed that T. gondii infection can alter the global metabolism of the cerebral cortex of mice. However, the impact of T. gondii infection on the metabolism of the cerebellum remains unknown. Here we apply metabolomic profiling to discover metabolic changes associated with T. gondii infection of the mouse cerebellum using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Multivariate statistics revealed differences in the metabolic profiles between the infected and control mouse groups and between the infected mouse groups as infection advanced. We also detected 10, 22, and 42 significantly altered metabolites (SAMs) in the infected cerebellum at 7, 14, and 21 days post infection (dpi), respectively. Four metabolites [tabersonine, arachidonic acid (AA), docosahexaenoic acid, and oleic acid] were identified as potential biomarker or responsive metabolites to T. gondii infection in the mouse cerebellum. Three of these metabolites (AA, docosahexaenoic acid, and oleic acid) play roles in the regulation of host behavior and immune response. Pathway analysis showed that T. gondii infection of the cerebellum involves reprogramming of amino acid and lipid metabolism. These results showcase temporal metabolomic changes during cerebellar infection by T. gondii in mice. The study provides new insight into the neuropathogenesis of T. gondii infection and reveals new metabolites and pathways that mediate the interplay between T. gondii and the mouse cerebellum.

Analysis of the serum peptidome associated with Toxoplasma gondii infection.

Toxoplasma gondii is an obligate intracellular parasite that can cause severe disease in immunocompromised individuals and congenitally infected neonates. In order to determine whether serum peptide profile could reveal disease markers or allow determination of toxoplasmosis aggressiveness, mouse sera were collected from acutely infected, chronically infected and control subjects, and analyzed by a quantitative label-free pepdomics approach (LC-MS/MS). Six hundred and seven endogenous peptides were identified among all samples, with peptide profiling of difference that readily distinguished between acutely infected samples and other samples. Among these peptides detected in this study, 81 and 68 differentially expressed peptides (DEPs) were found in the acute and chronic infection stages, respectively. Through Gene Ontology analysis, most of the precursor proteins of these DEPs were associated with biological regulation and binding activity. These findings in this study will help in the search of peptide targets with a key role in disease diagnosis and create new opportunities for the development of better means for the prevention and control of toxoplasmosis. SIGNIFICANCE: Toxoplasma gondii is an unicellular parasite which infects humans and a wide range of warm-blooded animals. The serum peptidome contains a large set of low molecular weight endogenous peptides derived from secretion, protease activity and PTMs. In the present study we quantified the effects of T. gondii infection on the serum peptidome to identify novel disease regulated secretory factors. We developed an optimized label-free LC-MS/MS method to analyze endogenous peptides during toxoplasmosis progression. This resulted in quantification of 607 unique peptides at both acute and chronic infection stages. Collectively, our deep peptidomic analysis of serum revealed that peptide variations were affected by disease development, and peptidomics is an ideal method for quantifying changes in circulating factors on a global scale in response to pathophysiological perturbations such as T. gondii infection.

miRNA and circRNA expression patterns in mouse brain during toxoplasmosis development.

BACKGROUND: Increasing evidence has shown that circular RNAs (circRNAs) are involved in neurodegenerative disorders, but their roles in neurological toxoplasmosis are yet to know. This study examined miRNA and circRNA expressions in mouse brain following oral infection with T. gondii Pru strain. RESULTS: Total RNA extracted from acutely infected (11 days post infection (DPI)), chronically infected (35 DPI) and uninfected mouse brain samples were subjected to genome-wide small RNA sequencing. In the acutely infected mice, 9 circRNAs and 20 miRNAs were upregulated, whereas 67 circRNAs and 28 miRNAs were downregulated. In the chronically infected mice, 2 circRNAs and 42 miRNAs were upregulated, whereas 1 circRNA and 29 miRNAs were downregulated. Gene ontology analysis predicted that the host genes that produced the dysregulated circRNAs in the acutely infected brain were primarily involved in response to stimulus and ion binding activities. Furthermore, predictive interaction networks of circRNA-miRNA and miRNA-mRNA were constructed based on genome-wide transcriptome sequencing and computational analyses, which might suggest the putative functions of miRNAs and circRNAs as a large class of post-transcriptional regulators. CONCLUSIONS: These findings will shed light on circRNA-miRNA interactions during the pathogenesis of toxoplasmosis, and they will lay solid foundation for studying the potential regulation roles of miRNAs and circRNAs in T. gondii induced pathogenesis.

Molecular Detection of Toxoplasma gondii in the Slaughter Sheep and Goats from Shandong Province, Eastern China.

It is generally recognized that sheep are susceptible to Toxoplasma gondii and play a very important role in the transmission of toxoplasmosis to humans. In China, sheep toxoplasmosis has been reported in some regions based on serological investigations. However, little is known about sheep toxoplasmosis in Shandong province, eastern China. Thus, this study was conducted to investigate the prevalence of T. gondii infection in the slaughter sheep and goats from three cities (Weihai, Yantai, and Rizhao) of Shandong province, eastern China. From November 2016 to March 2018, a total of 692 meat samples (438 sheep and 254 goats) were collected and detected by a seminested PCR-targeted T. gondii B1 gene. The overall prevalence of T. gondii in sheep and goats were 9.84% and 10.73%, respectively. Meat collected from rural markets (16.04%) had a significantly higher T. gondii prevalence than those collected from supermarkets (6.84%) (p < 0.001). Moreover, sheep and goats raised in backyard were more easily to be infected by T. gondii compared with those raised in farms (p < 0.001). This is the first report of the molecular prevalence of T. gondii infection in sheep and goats in Shandong province, eastern China, which would provide effective data for prevention and control of sheep and human toxoplasmosis in China.

Label-Free Quantitative Acetylome Analysis Reveals Toxoplasma gondii Genotype-Specific Acetylomic Signatures.

Distinct genotypic and pathogenic differences exist between Toxoplasma gondii genotypes. For example, genotype I is highly virulent, whereas genotype II and genotype III are less virulent. Moreover, Chinese 1 genotype (ToxoDB#9) is also virulent. Here, we compare the acetylomes of genotype 1 (RH strain) and Chinese 1 genotype (ToxoDB#9, PYS strain) of T. gondii. Using mass spectrometry enriched for acetylated peptides, we found a relationship between the levels of protein acetylation and parasite genotype-specific virulence. Notably, lysine acetylation was the largest (458 acetylated proteins) in RH strain, followed by PYS strain (188 acetylated proteins), whereas only 115 acetylated proteins were detected in PRU strain. Our analysis revealed four, three, and four motifs in RH strain, PRU strain and PYS strain, respectively. Three conserved sequences around acetylation sites, namely, xxxxxKAcHxxxx, xxxxxKAcFxxxx, and xxxxGKAcSxxxx, were detected in the acetylome of the three strains. However, xxxxxKAcNxxxx (asparagine) was found in RH and PYS strains but was absent in PRU strain. Our analysis also identified 15, 3, and 26 differentially expressed acetylated proteins in RH strain vs. PRU strain, PRU strain vs. PYS strain and PYS strain vs. RH strain, respectively. KEGG pathway analysis showed that a large proportion of the acetylated proteins are involved in metabolic processes. Pathways for the biosynthesis of secondary metabolites, biosynthesis of antibiotics and microbial metabolism in diverse environments were featured in the top five enriched pathways in all three strains. However, acetylated proteins from the virulent strains (RH and PYS) were more enriched in the pyruvate metabolism pathway compared to acetylated proteins from PRU strain. Increased levels of histone-acetyl-transferase and glycyl-tRNA synthase were detected in RH strain compared to PRU strain and PYS strain. Both enzymes play roles in stress tolerance and proliferation, key features in the parasite virulence. These findings reveal novel insight into the acetylomic profiles of major T. gondii genotypes and provide a new important resource for further investigations of the roles of the acetylated parasite proteins in the modulation of the host cell response to the infection of T. gondii.

iTRAQ-Based Global Phosphoproteomics Reveals Novel Molecular Differences Between Toxoplasma gondii Strains of Different Genotypes.

To gain insights into differences in the virulence among T. gondii strains at the post-translational level, we conducted a quantitative analysis of the phosphoproteome profile of T. gondii strains belonging to three different genotypes. Phosphopeptides from three strains, type I (RH strain), type II (PRU strain) and ToxoDB#9 (PYS strain), were enriched by titanium dioxide (TiO2) affinity chromatography and quantified using iTRAQ technology. A total of 1,441 phosphopeptides, 1,250 phosphorylation sites and 759 phosphoproteins were detected. In addition, 392, 298, and 436 differentially expressed phosphoproteins (DEPs) were identified in RH strain when comparing RH/PRU strains, in PRU strain when comparing PRU/PYS strains, and in PYS strain when comparing PYS/RH strains, respectively. Functional characterization of the DEPs using GO, KEGG, and STRING analyses revealed marked differences between the three strains. In silico kinase substrate motif analysis of the DEPs revealed three (RxxS, SxxE, and SxxxE), three (RxxS, SxxE, and SP), and five (SxxE, SP, SxE, LxRxxS, and RxxS) motifs in RH strain when comparing RH/PRU strains, in PRU strain when comparing PRU/PYS, and in PYS strain when comparing PYS/RH strains, respectively. This suggests that multiple overrepresented protein kinases including PKA, PKG, CKII, IKK, and MAPK could be involved in such a difference between T. gondii strains. Kinase associated network analysis showed that ROP5, ROP16, and cell-cycle-associated protein kinase CDK were the most connected kinase peptides. Our data reveal significant changes in the abundance of phosphoproteins between T. gondii genotypes, which explain some of the mechanisms that contribute to the virulence heterogeneity of this parasite.

Sulfadiazine Sodium Ameliorates the Metabolomic Perturbation in Mice Infected with Toxoplasma gondii.

In this study, we analyzed the global metabolomic changes associated with Toxoplasma gondii infection in mice in the presence or absence of sulfadiazine sodium (SDZ) treatment. BALB/c mice were infected with T. gondii GT1 strain and treated orally with SDZ (250 µg/ml in water) for 12 consecutive days. Mice showed typical manifestations of illness at 20 days postinfection (dpi); by 30 dpi, 20% had survived and developed latent infection. We used ultraperformance liquid chromatography-mass spectrometry to profile the serum metabolomes in control (untreated and uninfected) mice, acutely infected mice, and SDZ-treated and infected mice. Infection induced significant perturbations in the metabolism of α-linolenic acid, purine, pyrimidine, arginine, tryptophan, valine, glycerophospholipids, and fatty acyls. However, treatment with SDZ seemed to alleviate the serum metabolic alterations caused by infection. The restoration of the serum metabolite levels in the treated mice was associated with better clinical outcomes. These data indicate that untargeted metabolomics can reveal biochemical pathways associated with restoration of the metabolic status of T. gondii-infected mice following SDZ treatment and could be used to monitor responses to SDZ treatment. This study provides a new systems approach to elucidate the metabolic and therapeutic effects of SDZ in the context of murine toxoplasmosis.

Metabolomic signature of mouse cerebral cortex following Toxoplasma gondii infection.

BACKGROUND: The protozoan parasite Toxoplasma gondii infects and alters the neurotransmission in cerebral cortex and other brain regions, leading to neurobehavioral and neuropathologic changes in humans and animals. However, the molecules that contribute to these changes remain largely unknown. METHODS: We have investigated the impact of T. gondii infection on the overall metabolism of mouse cerebral cortex. Mass-spectrometry-based metabolomics and multivariate statistical analysis were employed to discover metabolomic signatures that discriminate between cerebral cortex of T. gondii-infected and uninfected control mice. RESULTS: Our results identified 73, 67 and 276 differentially abundant metabolites, which were involved in 25, 37 and 64 pathways at 7, 14 and 21 days post-infection (dpi), respectively. Metabolites in the unsaturated fatty acid biosynthesis pathway were upregulated as the infection progressed, indicating that T. gondii induces the biosynthesis of unsaturated fatty acids to promote its own growth and survival. Some of the downregulated metabolites were related to pathways, such as steroid hormone biosynthesis and arachidonic acid metabolism. Nine metabolites were identified as T. gondii responsive metabolites, namely galactosylsphingosine, arachidonic acid, LysoSM(d18:1), L-palmitoylcarnitine, calcitetrol, 27-Deoxy-5b-cyprinol, L-homophenylalanine, oleic acid and ceramide (d18:1/16:0). CONCLUSIONS: Our data provide novel insight into the dysregulation of the metabolism of the mouse cerebral cortex during T. gondii infection and have important implications for studies of T. gondii pathogenesis.

Acute Toxoplasma Gondii Infection in Cats Induced Tissue-Specific Transcriptional Response Dominated by Immune Signatures.

RNA-sequencing was used to detect transcriptional changes in six tissues of cats, seven days after T. gondii infection. A total of 737 genes were differentially expressed (DEGs), of which 410 were up-regulated and 327 were down-regulated. The liver exhibited 151 DEGs, lung (149 DEGs), small intestine (130 DEGs), heart (123 DEGs), brain (104 DEGs), and spleen (80 DEGs)-suggesting tissue-specific transcriptional patterns. Gene ontology and KEGG analyses identified DEGs enriched in immune pathways, such as cytokine-cytokine receptor interaction, Jak-STAT signaling pathway, NOD-like receptor signaling pathway, NF-kappa B signaling pathway, MAPK signaling pathway, T cell receptor signaling pathway, and the cytosolic DNA sensing pathway. C-X-C motif chemokine 10 (CXCL10) was involved in most of the immune-related pathways. PI3K/Akt expression was down-regulated in all tissues, except the spleen. The genes for phosphatase, indoleamine 2,3-dioxygenase, Hes Family BHLH Transcription Factor 1, and guanylate-binding protein 5, playing various roles in immune defense, were co-expressed across various feline tissues. Multivariate K-means clustering analysis produced seven gene clusters featuring similar gene expression patterns specific to individual tissues, with lung tissue cluster having the largest number of DEGs. These findings suggest the presence of a broad immune defense mechanism across various tissues in cats against acute T. gondii infection.

Hepatic Metabolomics Investigation in Acute and Chronic Murine Toxoplasmosis.

Toxoplasma gondii poses a great threat to human health, with no approved vaccine available for the treatment of T. gondii infection. T. gondii infections are not limited to the brain, and may also affect other organs especially the liver. Identification of host liver molecules or pathways involved in T. gondii replication process may lead to the discovery of novel anti-T. gondii targets. Here, we analyzed the metabolic profile of the liver of mice on 11 and 30 days postinfection (dpi) with type II T. gondii Pru strain. Global metabolomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 389 significant metabolites from acutely infected mice; and 368 from chronically infected mice, when compared with control mice. Multivariate statistical analysis revealed distinct metabolic signatures from acutely infected, chronically infected and control mice. Infection influenced several metabolic processes, in particular those for lipids and amino acids. Metabolic pathways, such as steroid hormone biosynthesis, primary bile acid biosynthesis, bile secretion, and biosynthesis of unsaturated fatty acids were perturbed during the whole infection process, particularly during the acute stage of infection. The present results provide insight into hepatic metabolic changes that occur in BALB/c mice during acute and chronic T. gondii infection.

Toxoplasma gondii and Neospora caninum in Tolai Hares (Lepus tolai) Intended for Human Consumption in China: Seroprevalence, DNA Detection, and Genotyping.

Currently, there is no information available on the detection of Toxoplasma gondii and Neospora caninum in the tissues of Tolai hares in China. This study aimed to investigate the prevalence of these protozoan parasites in Tolai hares obtained from Shandong province, eastern China, between January 2016 and June 2017. Serum and brain tissue samples of 358 Tolai hares were obtained and detected for the presence of antibody and parasite DNAs by serodiagnosis and polymerase chain reaction (PCR), respectively. The seroprevalence of T. gondii and N. caninum infection in Tolai hares was 8.10% (29/358) and 0.84% (3/358), respectively. However, all the 358 tested Tolai hares were negative for N. caninum by PCR and T. gondii DNA was detected in 23 Tolai hares (6.42%, 23/358). The positive T. gondii DNA was genotyped at 11 genetic markers using multilocus PCR-restriction fragment length polymorphism technology. Of the 23 positive samples, only 2 of them produced complete genotyping results, and were identified as ToxoDB Genotype #9. This is the first report to detect T. gondii in the tissues of Tolai hares from China and the first study to focus on N. caninum in Tolai hares from China.