The Toxoplasma protein phosphatase 6 catalytic subunit (TgPP6C) is essential for cell cycle progression and virulence.

Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii.

The antioxidant protein glutaredoxin 1 is essential for oxidative stress response and pathogenicity of Toxoplasma gondii.

Glutaredoxins (Grxs) are ubiquitous antioxidant proteins involved in many molecular processes to protect cells against oxidative damage. Here, we study the roles of Grxs in the pathogenicity of Toxoplasma gondii. We show that Grxs are localized in the mitochondria (Grx1), cytoplasm (Grx2), and apicoplast (Grx3, Grx4), while Grx5 had an undetectable level of expression. We generated Δgrx1-5 mutants of T. gondii type I RH and type II Pru strains using CRISPR-Cas9 system. No significant differences in the infectivity were detected between four Δgrx (grx2-grx5) strains and their respective wild-type (WT) strains in vitro or in vivo. Additionally, no differences were detected in the production of reactive oxygen species, total antioxidant capacity, superoxide dismutase activity, and sensitivity to external oxidative stimuli. Interestingly, RHΔgrx1 or PruΔgrx1 exhibited significant differences in all the investigated aspects compared to the other grx2-grx5 mutant and WT strains. Transcriptome analysis suggests that deletion of grx1 altered the expression of genes involved in transport and metabolic pathways, signal transduction, translation, and obsolete oxidation-reduction process. The data support the conclusion that grx1 supports T. gondii resistance to oxidative killing and is essential for the parasite growth in cultured cells and pathogenicity in mice and that the active site CGFS motif was necessary for Grx1 activity.

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.

Effect of deleting four Toxoplasma gondii calcium-binding EGF domain-containing proteins on parasite replication and virulence.

Several calcium-binding proteins including calcium-dependent protein kinases play important roles in several facets of the intracellular infection cycle of the apicomplexan protozoan parasite Toxoplasma gondii. However, the role of the calcium-binding epidermal growth factor (EGF) domain-containing proteins (CBDPs) remains poorly understood. In this study, we examined the functions of four CBDP genes in T. gondii RH strain of type I by generating knock-out strains using CRISPR-Cas9 system. We investigated the ability of mutant strains deficient in CBDP1, CBDP2, CBDP3, or CBDP4 to form plaques, replicate intracellularly, and egress from the host cells. The results showed that no definite differences between any of these four CBDP mutant strains and the wild-type strain in terms of their ability to form plaques, intracellular replication, and egress. Additionally, CBDP mutants did not exhibit any significant attenuated virulence compared to the wild-type strain in mice. The expression profiles of CBDP2-4 genes were conserved among T. gondii strains of different genotypes, life cycle stages, and developmental forms. Whether other CBDP genes play any roles in the pathogenicity of T. gondii strains of different genotypes remains to be elucidated.

Epidemiology, Pathophysiology, Diagnosis, and Management of Cerebral Toxoplasmosis.

Toxoplasma gondii is known to infect a considerable number of mammalian and avian species and a substantial proportion of the world's human population. The parasite has an impressive ability to disseminate within the host's body and employs various tactics to overcome the highly regulatory blood-brain barrier and reside in the brain. In healthy individuals, T. gondii infection is largely tolerated without any obvious ill effects. However, primary infection in immunosuppressed patients can result in acute cerebral or systemic disease, and reactivation of latent tissue cysts can lead to a deadly outcome. It is imperative that treatment of life-threatening toxoplasmic encephalitis is timely and effective. Several therapeutic and prophylactic regimens have been used in clinical practice. Current approaches can control infection caused by the invasive and highly proliferative tachyzoites but cannot eliminate the dormant tissue cysts. Adverse events and other limitations are associated with the standard pyrimethamine-based therapy, and effective vaccines are unavailable. In this review, the epidemiology, economic impact, pathophysiology, diagnosis, and management of cerebral toxoplasmosis are discussed, and critical areas for future research are highlighted.

Toxoplasma gondii tkl1 Deletion Mutant Is a Promising Vaccine against Acute, Chronic, and Congenital Toxoplasmosis in Mice.

In this study, we generated a tkl1 deletion mutant in the Toxoplasma gondii type 1 RH (RHΔtkl1) strain and tested the protective efficacies of vaccination using RHΔtkl1 tachyzoites against acute, chronic, and congenital T. gondii infections in Kunming mice. Mice vaccinated with RHΔtkl1 mounted a strong humoral and cellular response as shown by elevated levels of anti-T. gondii-specific IgG, IL-2, IL-12, IFN-γ, and IL-10. All RHΔtkl1-vaccinated mice survived a lethal challenge with 1 × 103 tachyzoites of type 1 RH or ToxoDB#9 (PYS or TgC7) strain as well as 100 cysts or oocysts of Prugniuad strain. All mock-vaccinated plus infected mice have died. Vaccination also protected against cyst- or oocyst-caused chronic infection, reduced vertical transmission caused by oocysts, increased litter size, and maintained body weight of pups born to dams challenged with 10 oocysts on day 5 of gestation. In contrast, all mock-vaccinated plus oocysts-infected dams had aborted, and no fetus has survived. Vaccinated dams remained healthy postinfection, and their brain cyst burden was significantly reduced compared with mock-vaccinated dams infected with oocysts. In vivo depletion of CD4+ T cells, CD8+ T cells, and B cells revealed that CD8+ T cells are involved in the protection of mice against T. gondii infection. Additionally, adoptive transfer of CD8+ T cells from RHΔtkl1-vaccinated mice significantly enhanced the survival of naive mice infected with the pathogenic strain. Together, these data reaffirm the importance of CD8+ T cell responses in future vaccine design for toxoplasmosis and present T. gondii tkl1 gene as a promising vaccine candidate.

Global phosphoproteome analysis reveals significant differences between sporulated oocysts of virulent and avirulent strains of Toxoplasma gondii.

In this study, the differences in the phosphoproteomic landscape of sporulated oocysts between virulent and avirulent strains of Toxoplasma gondii were examined using a global phosphoproteomics approach. Phosphopeptides from sporulated oocysts of the virulent PYS strain (Chinese ToxoDB#9) and the avirulent PRU strain (type II) were enriched by titanium dioxide (TiO2) affinity chromatography and quantified using IBT approach. A total of 10,645 unique phosphopeptides, 8181 nonredundant phosphorylation sites and 2792 phosphoproteins were identified. We also detected 4129 differentially expressed phosphopeptides (DEPs) between sporulated oocysts of PYS strain and PRU strain (|log1.5 fold change| > 1 and p < 0.05), including 2485 upregulated and 1644 downregulated phosphopeptides. Motif analysis identified 24 motifs from the upregulated phosphorylated peptides including 22 serine motifs and two threonine motifs (TPE and TP), and 15 motifs from the downregulated phosphorylated peptides including 12 serine motifs and three threonine motifs (TP, RxxT and KxxT) in PYS strain when comparing PYS strain to PRU strain. Several kinases were consistent with motifs of overrepresented phosphopeptides, such as PKA, PKG, CKII, IKK, MAPK, EGFR, INSR, Jak, Syk, Src, Ab1. GO enrichment, KEGG pathway analysis and STRING analysis revealed DEPs significantly enriched in many biological processes and pathways. Kinase related network analysis showed that AGC kinase was the most connected kinase peptide. Our findings reveal significant difference in phosphopeptide profiles of sporulated oocysts between virulent and avirulent T. gondii strains, providing new resources for further elucidation of the mechanisms underpinning the virulence of T. gondii.

Novel roles of dense granule protein 12 (GRA12) in Toxoplasma gondii infection.

Dense granule protein 12 (GRA12) is implicated in a range of processes related to the establishment of Toxoplasma gondii infection, such as the formation of the intravacuolar network (IVN) within the parasitophorous vacuole (PV). This protein is also thought to be important for T. gondii-host interaction, pathogenesis, and immune evasion, but their exact roles remain unknown. In this study, the contributions of GRA12 to the molecular pathogenesis of T. gondii infection were examined in vitro and in vivo. Deletion of GRA12 in type I RH and type II Pru T. gondii strains did not affect the parasite growth and replication in vitro, however, it caused a significant reduction in the parasite virulence and tissue cyst burden in vivo. T. gondii Δgra12 mutants were more vulnerable to be eliminated by host immunity, without the accumulation of immunity-related GTPase a6 (Irga6) onto the PV membrane. The ultrastructure of IVN in Δgra12 mutants appeared normal, suggesting that GRA12 is not required for biogenesis of the IVN. Combined deletion of GRA12 and ROP18 induced more severe attenuation of virulence compared to single Δgra12 or Δrop18 mutant strains. These data suggest a functional association between GRA12 and ROP18 that is revealed by the severe attenuation of virulence in a double mutant relative to the single individual mutations. Future studies are needed to define the molecular basis of this putative association. Collectively these findings indicate that although GRA12 is not essential for the parasite growth and replication in vitro, it contributes to the virulence and growth of T. gondii in mice.

Toxoplasma gondii induces metabolic disturbances in the hippocampus of BALB/c mice.

Toxoplasma gondii can cross the blood-brain barrier and infect different regions of the brain including the hippocampus. In the present study, we examined the impact of Toxoplasma gondii infection on the metabolism of the hippocampus of female BALB/c mice compared to control mice using ultra-high-performance liquid chromatography-tandem mass spectrometry. Multivariate analysis revealed significant differences between infected and control hippocampi and identified 25, 82, and 105 differential metabolites (DMs) in the infected hippocampi at 7, 14, and 21 days post-infection (dpi), respectively. One DM (sphingosyl-phosphocholine in the sphingolipid metabolism pathway) and 11 dysregulated pathways were detected at all time points post-infection, suggesting their important roles in the neuropathogenesis of T. gondii infection. These pathways were related to neural activity, such as inflammatory mediator regulation of TRP channels, retrograde endocannabinoid signaling, and arachidonic acid metabolism. Weighted correlation network analysis and receiver operating characteristic analysis identified 33 metabolites significantly associated with T. gondii infection in the hippocampus, and 30 of these were deemed as potential biomarkers for T. gondii infection. This study provides, for the first time, a global view of the metabolic perturbations that occur in the mouse hippocampus during T. gondii infection. The potential relevance of the identified metabolites and pathways to the pathogenesis of cognitive impairment and psychiatric disorders are discussed.

Lysine crotonylation is widespread on proteins of diverse functions and localizations in Toxoplasma gondii.

Lysine crotonylation (Kcr) is an evolutionally conserved post-translational modification (PTM) on histone proteins. However, information about Kcr and its involvement in the biology and metabolism of Toxoplasma gondii is limited. In the present study, a global Kcr proteome analysis using LC-MS/MS in combination with immune-affinity method was performed. A total of 12,152 Kcr sites distributed over 2719 crotonylated proteins were identified. Consistent with lysine acetylation and succinylation in Apicomplexa, Kcr was associated with various metabolic pathways, including carbon metabolism, pyrimidine metabolism, glycolysis, gluconeogenesis, and proteasome. Markedly, many stage-specific proteins, histones, and histone-modifying enzymes related to the stage transition were found to have Kcr sites, suggesting a potential involvement of Kcr in the parasite stage transformation. Most components of the apical secretory organelles were identified as crotonylated proteins which were associated with the attachment, invasion, and replication of T. gondii. These results expanded our understanding of Kcr proteome and proposed new hypotheses for further research of the Kcr roles in the pathobiology of T. gondii infection.

Characterization of functions in parasite growth and virulence of four Toxoplasma gondii genes involved in lipid synthesis by CRISPR-Cas9 system.

Fatty acid uptake is extremely important for the survival and growth of the intracellular parasite Toxoplasma gondii. In this study, CRISPR-Cas9 gene editing technology was used to investigate the role of four lipid synthesis enzymes, namely, glycerol-3-phosphate dehydrogenase (G3PDH), malonyl CoA-acyl carrier protein transacylase (FabD), acyl-ACP thiolesterase (TE), and diacylglycerol acyltransferase (DGAT), in the virulence and infectivity of Type I RH and Type II Prugniaud (Pru) strains of T. gondii. Immunofluorescence analysis of the tachyzoite stage showed that FabD protein was located in the apicoplast; however, the expression level of the other three proteins was undetectable. Compared with wild-type (WT) strains, the growth of RHΔG3PDH, RHΔTE, and RHΔDGAT in vitro and their virulence in vivo were not significantly different. However, RHΔFabD exhibited a significantly reduced growth rate, compared with the WT strain. The deletion of FabD attenuated the virulence of Type II Pru strain and reduced the formation of cysts in vivo. These data improved our understanding of the role of lipid synthesis enzymes in the pathogenesis of T. gondii.

Prevalence, genotypes and risk factors for Toxoplasma gondii contamination in marine bivalve shellfish in offshore waters in eastern China.

We conducted a large-scale epidemiological investigation to detect the prevalence of Toxoplasma gondii in four marine bivalve shellfish species collected from six representative coastal regions of Weihai, eastern China. Between January 2018 and December 2018, 14,535 marine bivalve shellfish pooled into 2907 samples were randomly collected and examined for T. gondii DNA by a nested PCR assay targeting B1 gene. The results showed that 2.8% (82) of the 2907 pooled samples were tested positive for T. gondii DNA. Two T. gondii genotype (ToxoDB Genotype #9 and ToxoDB Genotype #1) were identified PCR-restriction fragment length polymorphism analysis. Factors that were found significantly associated with the presence of T. gondii DNA in marine bivalve shellfish included the source of samples (being wild) (odds ratio [OR], 3.34; 95% confidence interval [CI], 2.00-5.84; p < 0.01), surface runoff near the sampling site (OR, 2.64; 95% CI, 1.47-4.72; p < 0.01), and presence of cats near the sampling site (OR, 1.77; 95% CI, 1.02-3.07; p = 0.04). Moreover, the prevalence of T. gondii DNA in marine bivalve shellfish correlated with temperature (Pearson's correlation: R = 0.75, p = 0.0049) and precipitation (R = 0.87, p = 0.00021). These findings provide new insights into the presence of T. gondii DNA in marine bivalve shellfish and highlight the impact of human activity on marine pollution by such an important terrestrial pathogen pollutant.

Development of a Lateral Flow Strip-Based Recombinase Polymerase Amplification Assay for the Detection of Haemonchus contortus in Goat Feces.

Haemonchosis remains a significant problem in small ruminants. In this study, the assay of recombinase polymerase amplification (RPA) combined with the lateral flow strip (LFS-RPA) was established for the rapid detection of Haemonchus contortus in goat feces. The assay used primers and a probe targeting a specific sequence in the ITS-2 gene. We compared the performance of the LFS-RPA assay to a PCR assay. The LFS-RPA had a detection limit of 10 fg DNA, which was 10 times less compared to the lowest detection limit obtained by PCR. Out of 24 goat fecal samples, LFS-RPA assay detected H. contortus DNA with 95.8% sensitivity, compared to PCR, 79.1% sensitivity. LFS-RPA assay did not detect DNA from other related helminth species and demonstrated an adequate tolerance to inhibitors present in the goat feces. Taken together, our results suggest that LFS-RPA assay had a high diagnostic accuracy for the rapid detection of H. contortus and merits further evaluation.

Biology, Epidemiology, Clinical Features, Diagnosis, and Treatment of Selected Fish-borne Parasitic Zoonoses.

Fish-borne parasites have been part of the global landscape of food-borne zoonotic diseases for many decades and are often endemic in certain regions of the world. The past 20 years or so have seen the expansion of the range of fish-borne parasitic zoonoses to new geographic regions leading to a substantial public health burden. In this article, we summarize current knowledge about the biology, epidemiology, clinical characteristics, diagnosis, treatment and control of selected fish-borne helminthic diseases caused by parasitic roundworm (Anisakis), tapeworm (Dibothriocephalus), and fluke (Metagonimus). Humans acquire infection via consumption of raw or improperly cooked fish or fish products. The burden from these diseases is caused by morbidity rather than mortality. Infected patients may present with mild to severe gastrointestinal (eg, abdominal pain, diarrhea, and indigestion) or allergic manifestations. Patients are often admitted to the hospital or clinic with acute symptoms and no prior health problems and no travel history. Diagnosis is often established based on the detection of the diagnostic parasite stages (eg, eggs or tapeworm segments) in the patient's feces. Sometimes imaging is required to exclude other causes and avoid unnecessary surgery. Dibothriocephalus and Metagonimus are mainly treated with praziquantel. Extraction of adult Dibothriocephalus or Anisakis larvae from the bowel ensures complete elimination of the parasites and prevents a relapse of infection. The development and implementation of more efficient food safety and public health strategies to reduce the burden of zoonotic diseases attributable to fish-borne parasites is highly desirable.

Immunostimulatory efficacy and protective potential of putative TgERK7 protein in mice experimentally infected by Toxoplasma gondii.

The extracellular signal-regulated kinases (ERKs) serve as important determinants of cellular signal transduction pathways, and hence may play important roles during infections. Previous work suggested that putative ERK7 of Toxoplasma gondii is required for efficient intracellular replication of the parasite. However, the antigenic and immunostimulatory properties of TgERK7 protein remain unknown. The objective of this study was to produce a recombinant TgERK7 protein in vitro and to evaluate its effect on the induction of humoral and T cell-mediated immune responses against T. gondii infection in BALB/c mice. Immunization using TgERK7 mixed with Freund's adjuvants significantly increased the ratio of CD3e+CD4+ T/CD3e+CD8a+ T lymphocytes in spleen and elevated serum cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-12p70, IL-23, MCP-1, and TNF-α) in immunized mice compared to control mice. On the contrary, immunization did not induce high levels of serum IgG antibodies. Five predicted peptides of TgERK7 were synthesized and conjugated with KLH and used to analyze the antibody specificity in the sera of immunized mice. We detected a progressive increase in the antibody level only against TgERK7 peptide A (DEVDKHVLRKYD). Antibody raised against this peptide significantly decreased intracellular proliferation of T. gondii in vitro, suggesting that peptide A can potentially induce a protective antibody response. We also showed that immunization improved the survival rate of mice challenged with a virulent strain and significantly reduced the parasite cyst burden within the brains of chronically infected mice. Our data show that TgERK7-based immunization induced TgERK7 peptide A-specific immune responses that can impart protective immunity against T. gondii infection. The therapeutic potential of targeting ERK7 signaling pathway for future toxoplasmosis treatment is warranted.

Global profiling of lysine 2-hydroxyisobutyrylome in Toxoplasma gondii using affinity purification mass spectrometry.

Lysine 2-hydroxyisobutyrylation (Khib) is a recently discovered and evolutionarily conserved form of protein post-translational modification (PTM) found in mammalian and yeast cells. Previous studies have shown that Khib plays roles in the activity of gene transcription and Khib-containing proteins are closely related to the cellular metabolism. In this study, a global Khib-containing analysis using the latest databases (ToxoDB 46, 8322 sequences, downloaded on April 16, 2020) and sensitive immune-affinity enrichment coupled with liquid chromatography-tandem mass spectrometry was performed. A total of 1078 Khib modification sites across 400 Khib-containing proteins were identified in tachyzoites of Toxoplasma gondii RH strain. Bioinformatics and functional enrichment analysis showed that Khib-modified proteins were associated with various biological processes, such as ribosome, glycolysis/gluconeogenesis, and central carbon metabolism. Interestingly, many proteins of the secretory organelles (e.g., microneme, rhoptry, and dense granule) that play roles in the infection cycle of T. gondii were found to be Khib-modified, suggesting the involvement of Khib in key biological process during T. gondii infection. We also found that histone proteins, key enzymes related to cellular metabolism, and several glideosome components had Khib sites. These results expanded our understanding of the roles of Khib in T. gondii and should promote further investigations of how Khib regulates gene expression and key biological functions in T. gondii.

Effect of deletion of gra17 and gra23 genes on the growth, virulence, and immunogenicity of type II Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii secretes a number of dense granule proteins (GRAs) from the dense granule organelle to manipulate the host cell. Two of these effector proteins (GRA17 and GRA23) are involved in the trafficking of molecules between the parasitophorous vacuole (PV) and the host cell cytoplasm. However, their roles in establishing chronic infection remain obscured. In this study, CRISPR-Cas9 was used to delete gra17 or gra23 gene in T. gondii Pru strain (type II). The growth, the virulence, the ability to establish chronic infection, and the immunogenicity of the constructed mutant strains were investigated in Kunming mice. Pru:Δgra17 and Pru:Δgra23 mutants developed PVs with abnormal morphology and exhibited reduced growth rate, compared with the wild-type Pru strain. Deletion of gra17 abrogated acute infection and blocked cyst formation. Although the deletion of gra23 caused slight attenuation of the parasite virulence in mice, it caused a significant reduction in cyst formation. Immunization with Pru:Δgra17 induced high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (interleukin-2 [IL-2], IL-10, IL-12, and interferon gamma [IFN-γ]), which conferred significant protection in mice challenged with virulent type I (RH), ToxoDB#9 (PYS) strains, or less virulent type II (Pru) strain of T. gondii. These findings show that GRA17 and GRA23 play important roles in T. gondii chronic infection and that irreversible deletion of gra17 in T. gondii type II Pru strain can be a viable option for stimulating protective immunity to T. gondii infection.

Acetylome analysis of the feline small intestine following Toxoplasma gondii infection.

Toxoplasma gondii is a protozoan parasite capable of infecting a large number of warm-blooded animals and causes serious health complications in immunocompromised patients. T. gondii infection of the feline small intestine is critical for the completion of the life cycle and transmission of T. gondii. Protein acetylation is an important posttranslational modification, which plays roles in the regulation of various cellular processes. Therefore, understanding of how T. gondii reprograms the protein acetylation status of feline definitive host can help to thwart the production and spread of T. gondii. Here, we used affinity enrichment and high-resolution liquid chromatography with tandem mass spectrometry to profile the alterations of the acetylome in cat small intestine 10 days after infection by T. gondii Prugniuad (Pru) strain. Our analysis showed that T. gondii induced significant changes in the acetylation of proteins in the cat intestine. We identified 2606 unique lysine acetylation sites in 1357 acetylated proteins. The levels of 334 acetylated peptides were downregulated, while the levels of 82 acetylated peptides were increased in the infected small intestine. The proteins with differentially acetylated peptides were particularly enriched in the bioenergetics-related processes, such as tricarboxylic acid cycle, oxidative phosphorylation, and oxidation-reduction. These results provide the first baseline of the global acetylome of feline small intestine following T. gondii infection and should facilitate further analysis of the role of acetylated protein in the pathogenesis of T. gondii infection in its definitive host.

Antioxidant enzymes as biomarkers of Cu and Pb exposure in the ground spiders Lycosa terrestris and Pardosa birmanica.

Heavy metal exposure induces oxidative stress in terrestrial organisms, which they counteract via activation of antioxidant biomarkers. The present study investigated the effects of copper (Cu) and lead (Pb) on the total antioxidant capacity (TAC) and antioxidant enzymes such as Catalase (CAT), Glutathione reductase (GR), Superoxide dismutase (SOD) and Glutathione peroxidase (GPX) in two spider species, namely Lycosa terrestris and Pardosa birmanica. The spiders were exposed to Cu and Pb separately (10 ppm) or in combination (10 ppm each) via two different exposure routes (i.e. food and soil) for 10, 20 and 40 days. The results showed that metal accumulation and antioxidant biomarker responses in spiders were metal- and species-dependent. Also, the levels of all antioxidant biomarkers increased significantly with increasing exposure time and metal load in the bodies of spiders via both exposure routes. The significant inhibition of TAC and antioxidant enzyme activities was only observed in single Pb treatment through soil exposure. In L. terrestris, the activities of detoxification enzymes and TAC were significantly enhanced on single Cu exposure than Pb via both exposure routes. However, in P. birmanica consistent variation among antioxidant parameters were observed depending on the metal load and exposure routes. The combined metal exposure caused more pronounced increase in the level of antioxidants compared to single metal exposure in both species, mainly via food exposure. These results suggest that the antioxidant enzymes and TAC are sensitive to single and combined metal exposure via both uptake routes. These data show that antioxidant parameters can be used potential biomarkers of oxidative stress associated with metal exposure and for monitoring environmental health using spiders as bioindicators.

Prevalence, risk factors and genotype distribution of Toxoplasma gondii DNA in soil in China.

In the present study, we performed a cross-sectional survey to determine the occurrence and genotype distribution of T. gondii DNA in soil samples collected from different sources from six geographic regions in China. Between March 2015 and June 2017, 2100 soil samples were collected from schools, parks, farms and coastal beaches, and examined for T. gondii DNA using three PCR assays targeting 529-bp repeat element (RE) sequence, B1 gene and ITS-1 gene sequences. Also, we investigated whether geographic region, soil source and type, and sampling season can influence the prevalence of T. gondii DNA in the soil. Soil samples collected from farms and parks had the highest prevalence, whereas samples collected from school playgrounds and coastal beaches had the lowest prevalence. PCR assays targeting 529-bp RE and ITS-1 gene sequences were more sensitive than the B1 gene-based assay. Positive PCR products were genotyped using multi-locus PCR-RFLP, and ToxoDB #9 was the predominant genotype found in the contaminated soil samples. Multiple logistic regression identified factors correlated significantly with the presence of T. gondii DNA in the soil to be the source of the soil, including farms (odds ratio 3.10; 95% confidence interval [CI], 1.52 to 6.29; p = 0.002) and parks (2.59; 95% CI 1.28 to 5.27; p = 0.009). These results show that Chinese soil hosts T. gondii of the most prevalent genotype in China (ToxoDB#9) and that the soil type influences infection patterns.