Potential Association Between Latent Toxoplasma gondii Infection and Suicide Attempts: A Case-Control Registry-Based Study.

Toxoplasma gondii is a ubiquitous parasitic protozoan that may be an important cause of neurological and psychiatric diseases. The purpose of this case-control registry-based study was to evaluate the prevalence of T. gondii infection and related risk factors among subjects who attempted suicide by drug use and a control group at the Iranian National Registry Center for Toxoplasmosis in Mazandaran Province, northern Iran. Baseline data were collected from participants using a questionnaire, and a blood sample was taken from each individual. The plasma was prepared for serological analysis, whereas the buffy coat was used for molecular analysis. Out of 282 individuals (147 cases with suicide attempters [SA] and 135 controls), 42.9% of patients and 16.3% of control subjects were positive for anti-Toxoplasma immunoglobin G (IgG), but all participants were negative for T. gondii DNA and anti-Toxoplasma immunoglobin M. Based on multiple logistic regressions, IgG seropositivity in SA in the age group of 20-30 years was 3.22 times higher than that in the control group (p < 0.001). These findings suggest that latent T. gondii infection among SA is significantly higher than that in healthy individuals, indicating a potential association between latent toxoplasmosis and SA at least in the studied area. Further research is needed to shed light on the potential association between T. gondii and suicide among different populations and areas of the world.

Phosphoproteomic analysis reveals changes in A-Raf-related protein phosphorylation in response to Toxoplasma gondii infection in porcine macrophages.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes severe threats to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important posttranslational modification involved in diverse cellular functions. A rapidly accelerated fibrosarcoma kinase (A-Raf) is a member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that knockout of A-Raf could reduce T. gondii-induced apoptosis in porcine alveolar macrophages (3D4/21 cells). However, limited information is available on protein phosphorylation variations and the role of A-Raf in macrophages infected with T. gondii. METHODS: We used immobilized metal affinity chromatography (IMAC) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile changes in phosphorylation in T. gondii-infected 3D4/21 and 3D4/21-ΔAraf cells. RESULTS: A total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in T. gondii-infected 3D4/21 cells (p3T group) when compared with uninfected 3D4/21 cells (pho3 group), and 959 DEPPs with 1540 DPSs were identified in the p3T group compared with infected 3D4/21-ΔAraf cells (p3KT group). Venn analysis revealed 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites when comparing p3T/pho3 versus p3T/p3KT, which were identified as DPSs and DEPPs that were directly or indirectly related to A-Raf. CONCLUSIONS: Our results revealed distinct responses of macrophages to T. gondii infection and the potential roles of A-Raf in fighting infection via phosphorylation of crucial proteins.

Serological Screening of TORCH Pathogen Infections in Infertile Women of Childbearing Age in Northwest China.

Serological screening for TORCH(Toxoplasma gondii [TOX], Rubella virus [RV], Cytomegalovirus [CMV], and Herpes simplex virus [HSV]) infections is an effective method for preventing congenital infections caused by TORCH pathogens.In this study, we retrospectively analyzed the characteristics of TORCH infections in 17,807 infertile women of childbearing age in northwest China.We conducted serological detection of TORCH-pathogen-specific IgM and IgG antibodies. The seroprevalence of TORCH infections was statistically analyzed by applying χ2 and Fisher exact-probability tests to evaluate the differences among ages and across quarters of the year. The overall IgM/IgG seroprevalences of TOX, RV, CMV, HSV-1, and HSV-2 were 0.46/3.4%, 0.77/84.93%, 0.68/97.54%, 1.2/82.83%, and 0.62/10.04%, respectively. The positive rates for RV-IgM in women ≥ 40 years old were significantly higher than those for women 25-39 (P < 0.05) years of age. The seroprevalence of HSV1-IgM was higher in the third and fourth quarters of the year (seasons) (P < 0.001), and the seroprevalence of CMV-IgG was statistically significant between differences quarters (P = 0.017), and the seroprevalence of CMV-IgG in the first quarter was lower than that in the third and fourth quarters (Bonferroni correction, P = 0.009 > 0.0083), suggesting no statistically significant difference between the latter two groups. This study showed that in northwestern China the risk of acquiring primary infection by a TORCH pathogen among infertile women of childbearing age were still high, especially Toxoplasma gondii and Herpesvirus type 2 infection. Therefore, effective prevention strategies that include serological screening for TORCH should be implemented.

The Crk4-Cyc4 complex regulates G2/M transition in Toxoplasma gondii.

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4-TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4-TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4-Cyc4 complex is limited to a minor group of the binary dividing apicomplexans.

Association between Toxoplasma gondii and gestational diabetes with regard to serum leptin and tumor necrosis factor alpha, a preliminary study.

Gestational diabetes (GDM), the onset of any degree of glucose intolerance during pregnancy, increases a wide range of adverse health outcomes for both the mother and the fetus. The aim of the present study was to evaluate the association of Toxoplasma gondii infection with GDM in a case-control study with regard to the levels of leptin and tumor necrosis factor alpha (TNF-α) as two inflammatory biomarkers. Fifty-one pregnant diabetic cases and 109 controls were selected from a prenatal care clinic of a general hospital in Shiraz, southern Iran during July-November 2020. Cases and controls were similar in age, gestational age and number of parturitions. The presence of IgG antibodies against T. gondii, and serum concentrations of leptin and TNF-α were determined by ELISA. Anti-Toxoplasma antibodies were detected in 25 subjects (15.6 %, 95 % CI: 9.9-21.3). Nine (18 %) diabetic cases were infected with Toxoplasma compared to 16 (15 %) healthy controls (P = 0.63). Level of leptin was higher (P = 0.07) while TNF-α was lower in diabetic cases compared to healthy controls (P = 0.08). When subjects were classified according to the combination of GDM and T. gondii, leptin was significantly lower in healthy (non-diabetic, non-infected) subjects compared to diabetics (P = 0.026), and TNF-α was higher in healthy subjects compared to Toxoplasma-infected diabetics (P = 0.032). These findings can be interpreted as both comorbidities being individually associated with increasing serum leptin and decreasing TNF-α concentrations, with modifying effects on each other. The present study opens a new perspective on GDM and its complex pathophysiological mechanism. Future research in this area is needed to better understand the underlying pathway for the development of GDM and the role of T. gondii and inflammatory biomarkers.

Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness.

BACKGROUND: To successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4. METHODS: We characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4. RESULTS: Trx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice. CONCLUSIONS: Trx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment.

Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin.

BACKGROUND: Toxoplasma gondii is an apicomplexan intracellular obligate parasite and the etiological agent of toxoplasmosis in humans, domestic animals and wildlife, causing miscarriages and negatively impacting offspring. During its intracellular development, it relies on nutrients from the host cell, controlling several pathways and the cytoskeleton. T. gondii has been proven to control the host cell cycle, mitosis and cytokinesis, depending on the time of infection and the origin of the host cell. However, no data from parallel infection studies have been collected. Given that T. gondii can infect virtually any nucleated cell, including those of humans and animals, understanding the mechanism by which it infects or develops inside the host cell is essential for disease prevention. Therefore, we aimed here to reveal whether this modulation is dependent on a specific cell type or host cell species. METHODS: We used only primary cells from humans and bovines at a maximum of four passages to ensure that all cells were counted with appropriate cell cycle checkpoint control. The cell cycle progression was analysed using fluorescence-activated cell sorting (FACS)-based DNA quantification, and its regulation was followed by the quantification of cyclin B1 (mitosis checkpoint protein). The results demonstrated that all studied host cells except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Additionally, we used an immunofluorescence assay to track mitosis and cytokinesis in uninfected and T. gondii-infected cells. RESULTS: The results demonstrated that all studied host cell except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Our findings showed that the analysed cells developed chromosome segregation problems and failed to complete cytokinesis. Also, the number of centrosomes per mitotic pole was increased after infection in all cell types. Therefore, our data suggest that T. gondii modulates the host cell cycle, chromosome segregation and cytokinesis during infection or development regardless of the host cell origin or type.

Seroprevalence and risk factors of Toxoplasma gondii infection in goats from South Punjab Province, Pakistan.

Layyah District in South Punjab Province of Pakistan offers the most intensive caprine economy in the country; its Indus riverine and desert environment makes the area peculiar and worthy of specific investigations. This study aimed to investigate the prevalence of anti-Toxoplasma gondii (T. gondii) IgG-antibody in goats in serum samples and the potential risk factors. The prevalence of T. gondii infection was estimated using a two-stage sample design. All caprine farms in the study area were stratified by size, and from these 110 were randomly selected. Twelve goats (>1-year-old) were selected from each farm and a total of 1320 serum samples were collected and tested by ELISA. A questionnaire on the conditions and management practices of each farm was administered to 110 farmers. Four hundred and sixteen out of 1320 sera samples (31.5%) were found positive and 89% of the flock had at least one seropositive goat. The proportion of seropositive goats tested within each flock ranged from 8.3% to 83.3%. with several factors contributing to this heterogeneity. Goat age played a significant role in the presence of cats. Significant interactions were related to goat farms having floor of dirt and kitten presence. Moreover, age class, abortion history and water source supply were modulated by owner education levels. This is the first study to determine the prevalence of anti-T. gondii antibodies in goats sera in Layyah district and the largest carried out so far in Pakistan. The remarkable presence of T. gondii among goats in areas where goat farming plays a significant economic role may pose a production threat to the small-stock industry, as well as to public health and food safety. Therefore, investigations to identify high-risk goat populations are highly recommended in order to facilitate the implementation of local control strategies.

Host cell-specific metabolism of linoleic acid controls Toxoplasma gondii growth in cell culture.

The obligate intracellular parasite Toxoplasma gondii can infect and replicate in any warm-blooded cell tested to date, but much of our knowledge about T. gondii cell biology comes from just one host cell type: human foreskin fibroblasts (HFFs). To expand our knowledge of host-parasite lipid interactions, we studied T. gondii in intestinal epithelial cells, the first site of host-parasite contact following oral infection and the exclusive site of parasite sexual development in feline hosts. We found that highly metabolic Caco-2 cells are permissive to T. gondii growth even when treated with high levels of linoleic acid (LA), a polyunsaturated fatty acid (PUFA) that kills parasites in HFFs. Caco-2 cells appear to sequester LA away from the parasite, preventing membrane disruptions and lipotoxicity that characterize LA-induced parasite death in HFFs. Our work is an important step toward understanding host-parasite interactions in feline intestinal epithelial cells, an understudied but important cell type in the T. gondii life cycle.

Alterations in gut microbiota contribute to cognitive deficits induced by chronic infection of Toxoplasma gondii.

Chronic infection with Toxoplasma gondii (T. gondii) emerges as a risk factor for neurodegenerative diseases in animals and humans. However, the underlying mechanisms are largely unknown. We aimed to investigate whether gut microbiota and its metabolites play a role in T. gondii-induced cognitive deficits. We found that T. gondii infection induced cognitive deficits in mice, which was characterized by synaptic ultrastructure impairment and neuroinflammation in the hippocampus. Moreover, the infection led to gut microbiota dysbiosis, barrier integrity impairment, and inflammation in the colon. Interestingly, broad-spectrum antibiotic ablation of gut microbiota attenuated the adverse effects of the parasitic infection on the cognitive function in mice; cognitive deficits and hippocampal pathological changes were transferred from the infected mice to control mice by fecal microbiota transplantation. In addition, the abundance of butyrate-producing bacteria and the production of serum butyrate were decreased in infected mice. Interestingly, dietary supplementation of butyrate ameliorated T. gondii-induced cognitive impairment in mice. Notably, compared to the healthy controls, decreased butyrate production was observed in the serum of human subjects with high levels of anti-T. gondii IgG. Overall, this study demonstrates that gut microbiota is a key regulator of T. gondii-induced cognitive impairment.

Indirect impact of SARS-CoV-2 pandemic on incidence of maternal primary cytomegalovirus and Toxoplasma gondii infection in pregnancy.

OBJECTIVE: Public health interventions promoted during the SARS-CoV-2 pandemic to control viral spread have impacted the occurrence of other communicable disease. Yet no studies have focused on perinatal infections with the potential for neonatal sequelae, including cytomegalovirus (CMV) and Toxoplasma gondii (TG). Here we investigate whether incidence rates of maternal primary CMV and TG infection in pregnancy were affected by the implementation of pandemic-related public health measures. METHODS: A retrospective study including all pregnant women with confirmed primary CMV or TG infection in pregnancy, managed between 2018 and 2021 at two university centers. The incidence rate was calculated as the number of CMV and TG infections per 100 consultations with a 95% confidence interval (CI). Data were compared between pre-pandemic (2018-2019) and pandemic (2020 and 2021) years. The Newcombe Wilson with Continuity Correction method was employed to compare incidence rates. RESULTS: The study population included 215 maternal primary CMV and 192 TG infections. Rate of maternal primary CMV infection decreased in 2021 compared with 2018-2019 (4.49% vs 6.40%, attributable risk [AR] 1.92, P = 0.019). By contrast, the rate of TG infection substantially increased in 2020 (6.95% vs 4.61%, AR 2.34, P = 0.006). Close contact with cats was more common among patients with TG infection in 2020 and 2021 than among pre-pandemic TG-infected women (26.3% and 24.4% vs 13.3%, P = 0.013). CONCLUSION: Pandemic-related public health interventions and associated behavioral and lifestyle changes exerted a divergent effect on the incidence of primary CMV and TG infection in pregnancy, likely due to modulation of exposure to risk factors for these infections.

Impact of latent toxoplasmosis on pneumonic and non-pneumonic COVID-19 patients with estimation of relevant oxidative stress biomarkers.

Susceptibility to COVID-19, the most devastating global pandemic, appears to vary widely across different population groups. Exposure to toxoplasmosis has been proposed as a theory to explain the diversity of these populations. The aim of the present study was to investigate the possible association between latent toxoplasmosis and COVID-19 and its probable correlation with markers of oxidative stress, C-reactive protein (CRP) and ferritin. In a case-control study, blood samples were collected from 91 confirmed (48 non-pneumonic; NP, and 43 pneumonic; P) COVID-19 patients and 45 healthy controls. All participants were tested for IgG anti-Toxoplasma gondii antibodies and oxidative stress markers (nitric oxide [NO], superoxide dismutase [SOD] and reduced glutathione [GSH]), and CRP and serum ferritin levels were determined. In COVID-19 patients, IgG anti-T. gondii antibodies were found in 54% compared to 7% in the control group, with the difference being statistically significant (P ˂ 0.001). However, no significant correlation was found between the severity of COVID-19 and latent T. gondii infection. Latent toxoplasmosis had a strong influence on the risk of COVID-19. NO and SOD levels were significantly increased in COVID-19 patients, while GSH levels decreased significantly in them compared to control subjects (P ˂ 0.001 for both values). CRP and ferritin levels were also significantly elevated in P COVID-19 patients infected with toxoplasmosis. This is the first study to look at the importance of oxidative stress indicators in co-infection between COVID-19 and T. gondii. The high prevalence of latent toxoplasmosis in COVID-19 suggests that T. gondii infection can be considered a strong indicator of the high risk of COVID-19.

Seromolecular study on the prevalence and risk factors of Toxoplasma gondii infection in pregnant women referred to a gynecology hospital in Urmia, northwest part of Iran in 2022.

Toxoplasmosis is a frequent infection among the human population. The infection can cause devastating complications for the fetus during pregnancy. The present study aimed to determine the serological and molecular prevalence of the infection and molecular characterization of Toxoplasma gondii isolates among pregnant women referred to Kowsar Hospital, Urmia, Iran. In a cross-sectional study, 340 blood samples were collected from pregnant women referred to Kowsar Hospital, Urmia, Iran from May to July 2022. Anti-T. gondii IgG and IgM seropositivity were determined by enzyme-linked immunosorbent assay. PCR was carried out by targeting the GRA6 gene of the parasite on all patients' buffy coats. Anti-T. gondii IgG and IgM antibodies were positive in two (0.6%) women, and 101 (29.7%) women had anti-T. gondii IgG and 70.3% were seronegative. PCR was positive in two IgM-positive women, and both isolates belonged to T. gondii carrying the GRA6 allele of lineage I. The risk of infection was significantly higher in women who had constant contact with cats and soil, and who were residents of rural areas. The two IgM-positive women were asymptomatic regarding acute toxoplasmosis. According to the results of the present study, the prevalence of toxoplasmosis in pregnant women in Urmia is similar to its prevalence in other areas in northwestern Iran, and despite the low prevalence of acute infection, it should not be ignored.

Inhibition of Foxp3 expression in the placenta of mice infected intraperitoneally by toxoplasma gondii tachyzoites: insights into the PPARγ/miR-7b-5p/Sp1 signaling pathway.

BACKGROUND: Toxoplasma gondii, an obligate intracellular parasitic protozoa, infects approximately 30% of the global population. Contracting T. gondii at the primary infection of the mother can result in neonatal microcephaly, chorioretinitis, hydrocephalus, or mortality. Our previous study indicated that pregnant mice infected with T. gondii displayed a decrease in both the number and the suppressive ability of regulatory T cells, accompanied by the reduced Forkhead box P3 (Foxp3). Numerous studies have proved that microRNAs (miRNAs) are implicated in T. gondii infection, but there is meager evidence on the relationship between alterations of miRNAs and downregulation of Foxp3 induced by T. gondii. METHODS: Quantitative reverse transcription polymerase chain reaction was utilized to detect the transcriptions of miRNAs and Foxp3. Protein blotting and immunofluorescence were used to detect the expressions of Foxp3 and related transcription factors. The structure of mouse placenta was observed by hematoxylin and eosin (HE) staining. To examine the activity of miR-7b promoter and whether miR-7b-5p targets Sp1 to suppress Foxp3 expression, we constructed recombinant plasmids containing the full-length/truncated/mutant miR-7b promoter sequence or wildtype/mutant of Sp1 3' untranslated region (3' UTR) to detect the fluorescence activity in EL4 cells. RESULTS: In T. gondii-infected mice, miR-7b transcription was significantly elevated, while Foxp3 expression was decreased in the placenta. In vitro, miR-7b mimics downregulated Foxp3 expression, whereas its inhibitors significantly upregulated Foxp3 expression. miR-7b promoter activity was elevated upon the stimulation of T. gondii antigens, which was mitigated by co-transfection of mutant miR-7b promoter lacking peroxisome proliferator-activated receptor γ (PPARγ) target sites. Additionally, miR-7b mimics diminished Sp1 expression, while miR-7b inhibitors elevated its expression. miR-7b mimics deceased the fluorescence activity of Sp1 3' untranslated region (3' UTR), but it failed to impact the fluorescence activity upon the co-transfection of mutant Sp1 3' UTR lacking miR-7b target site. CONCLUSIONS: T. gondii infection and antigens promote miR-7b transcription but inhibit Foxp3 protein and gene levels. T. gondii antigens promote miR-7b promoter activity by a PPARγ-dependent mechanism. miR-7b directly binds to Sp1 3' UTR to repress Sp1 expression. Understanding the regulatory functions by which T. gondii-induced miR-7b suppresses Foxp3 expression can provide new perspectives for the possible therapeutic avenue of T. gondii-induced adverse pregnancy outcomes.

Deciphering the epidemiological dynamics: Toxoplasma gondii seroprevalence in mainland China's food animals, 2010-2023.

Background: Toxoplasma gondii (T. gondii) is a significant protozoan pathogen among food animals. Despite the threat to public health by T. gondii infections, there's limited understanding of its seroprevalence and trends in food animals across mainland China. This study aimed to estimate the seroprevalence of T. gondii infections among swine, sheep, goats, chickens, and cattle in mainland China from 2010 to 2023. Methods: We searched cross-sectional studies published between 2010 and 2023 that reported the prevalence of T. gondii in food animals from databases including PubMed, Embase, Web of Science, China Biology Medicine Disc (CBM), China National Knowledge Infrastructure (CNKI), Wanfang data, and the China Science and Technology Journal Database (CQVIP). We performed subgroup analyses to explore the impact of different factors on the seroprevalence of T. gondii. Pooled estimates of T. gondii seroprevalence were calculated with a random-effects model. Results: An analysis of 184 studies involving 211985 animals revealed a T. gondii overall seroprevalence of 15.3% (95% CI: 13.1-17.8). Although the seroprevalence of food animals across mainland China was relatively stable from 2010 to 2023, notable variations were observed across different animal types and regions (P < 0.01), along with changes in geographical distribution. Sample type, detection method, animal age, and history of abortion were identified as key risk factors for T. gondii seroprevalence. Conclusion: The study conducted a meta-analysis on the seroprevalence of T. gondii in mainland China's Food Animals from 2010 to 2023, and identified key risk factors. These findings advance our understanding of T. gondii infection dynamics, offering critical insights for developing control strategies and guiding public health policies.

Evaluation of In Vitro Cytotoxic and Apoptotic Effects of Miltefosine on the Toxoplasma gondii RH Strain.

Background: We aimed to investigate the cytotoxic and apoptotic effects of miltefosine on Toxoplasma gondii RH strain by various techniques. Methods: The study was conducted at the Department of Parasitology and Mycology, Urmia University of Medical Sciences, Iran in 2020. Four groups of five BALB/c mice were selected. The cytotoxicity test was conducted by adding miltefosine to T. gondii tachyzoites; control tachyzoites received PBS and MTT assay was done on each suspension. For evaluating the Th1-type immune responses, the serum levels of IFN-γ and nitric oxide (NO) were assessed in mice after injecting tachyzoites and miltefosine, respectively. The flow cytometry technique was performed on T. gondii tachyzoites challenged with IC50 and IC90 doses of miltefosine and unchallenged cells. DNA fragments in T. gondii tachyzoites were detected by Terminal dUTPnick-end labeling (TUNEL) method. Results: Overall, 256, 64, 32, and 16 µg concentrations of miltefosine, respectively could kill more than 50% of viable T. gondii tachyzoites. The infected mice group, treated with miltefosine, significantly produced more IFN-γ relative to other groups (P< 0.001). Moreover, a significant difference was found in inducible NO synthase between the experimental and control groups (P<0.05). The flow cytometry results demonstrated a concentration-dependent apoptosis rate in tachyzoites incubated with miltefosine, though the necrosis rate was non-significant. DNA fragmentation analysis indicated oligonucleotides (18-200 bp) in tachyzoites treated with 11µg of miltefosine for 24, 48 and 72 h. However, this pattern was not observed in untreated control microorganisms. Conclusion: Miltefosine could be a favorable candidate for use as a new treatment for toxoplasmosis.

Toxoplasma gondii suppresses proliferation and migration of breast cancer cells by regulating their transcriptome.

BACKGROUND: Breast cancer is the most common cancer in women worldwide. Toxoplasma gondii (T. gondii) has shown anticancer activity in breast cancer mouse models, and exerted beneficial effect on the survival of breast cancer patients, but the mechanism was unclear. METHODS: The effect of tachyzoites of T. gondii (RH and ME49 strains) on human breast cancer cells (MCF-7 and MDA-MB-231 cells) proliferation and migration was assessed using cell growth curve and wound healing assays. Dual RNA-seq was performed for T. gondii-infected and non-infected cells to determine the differentially expressed genes (DEGs). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-Protein Interaction Networks analysis (PPI) were performed to explore the related signaling pathway and hub genes. Hub genes were validated using the Kaplan-Meier plotter database, and Pathogen Host Interaction (PHI-base) database. The results were verified by qRT-PCR. RESULTS: The tachyzoites of T. gondii decreased the expression of Ki67 and increased the expression of E-cadherin, resulting in suppressing the proliferation and migration of infected human breast cancer cells. The inhibitory effect of T. gondii on breast cancer cells showed a significant dose-response relationship. Compared with the control group, 2321 genes were transcriptionally regulated in MCF-7 cells infected with T. gondii, while 169 genes were transcriptionally regulated in infected MDA-MB-231 cells. Among these genes, 698 genes in infected MCF-7 cells and 67 genes in infected MDA-MB-231 cells were validated by the publicly available database. GO and KEGG analyses suggested that several pathways were involved in anticancer function of T. gondii, such as ribosome, interleukin-17 signaling, coronavirus disease pathway, and breast cancer pathway. BRCA1, MYC and IL-6 were identified as the top three hub genes in infected-breast cancer cells based on the connectivity of PPI analysis. In addition, after interacting with breast cancer cells, the expression of ROP16 and ROP18 in T. gondii increased, while the expression of crt, TgIST, GRA15, GRA24 and MIC13 decreased. CONCLUSIONS: T. gondii transcriptionally regulates several signaling pathways by altering the hub genes such as BRCA1, MYC and IL-6, which can inhibit the breast tumor growth and migration, hinting at a potential therapeutic strategy.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

Risk factors for Toxoplasma gondii infection in dogs: a serological survey.

BACKGROUND: Dogs, as well as a wide variety of other warm-blooded animals, act as intermediate host of Toxoplasma gondii. In dogs, most cases of toxoplasmosis are subclinical, although clinical disease has been sporadically reported. Beyond its role in diagnostic pathways, seropositivity also functions as a reflection of the parasite's spread within the dog's living environment. The aim of the present study was to evaluate the possible risk factor associated with seropositivity to T. gondii in dogs in Central-Northern Italy, analysing 120 dogs sera for the presence of IgG antibodies by indirect fluorescence antibody test (IFAT). RESULTS: The population examined was composed of 54.2% hunting dogs, 24.2% companion dogs, 14.2% truffle dogs and 7.5% watchdogs. Thirty-four (29.2%) dogs tested positive for T. gondii IgG, with titres ranging from 1:40 to 1:1280. Seroprevalence and antibodies titres were not related to dog gender, age or function. The logistic regression and ordered logistic regression results indicated that seroprevalence, and antibody titres were significantly higher in dogs cohabiting with cats, exhibiting coprophagy habits, and living constantly outdoors. Notably, the lifestyle factor showed the highest odds-ratios in the study: dogs living constantly outdoors were found to be at approximately 5 times greater risk of testing positive and having higher antibody titres compared to dogs living both indoors and outdoors. CONCLUSION: Both logistic and ordered logistic regression results support the key role of living with cats, engaging in coprophagy behaviours, and maintaining an outdoor lifestyle in increasing the risk of T. gondii infection in dogs. These identified risk factors collectively suggest that both ingesting oocysts, as observed through cat cohabitation and coprophagy, and engaging in predatory behaviours, as possible for outdoor living dogs, are indicating likely sources of T. gondii infection in this host species.

A systematic review and meta-analysis of the validation of serological methods for detecting anti-Toxoplasma gondii antibodies in humans and animals.

Toxoplasma gondii is a paradigmatic zoonotic parasite from the One Health perspective, since it is broadly distributed and virtually infects all warm-blooded species. A wide variety of serological techniques have been developed to detect T. gondii infection in humans and animals. Our aim was to describe and compare the main characteristics of these serological tests and validation processes and to critically analyze whether these tests meet the standards required to ensure an accurate serological diagnosis. The current systematic review and meta-analysis included 134 studies that were published from 2013 to 2023. QUADAS 2 tool was used to evaluate the quality of the included studies. A total of 52 variables related to the characteristics of the techniques and analytical and diagnostic validation parameters were studied. A wider panel of tests was developed for humans, including techniques exclusively developed for humans that involve costly equipment and the measurement of different Ig isotypes that are considered biomarkers of congenital toxoplasmosis. Studies conducted in humans frequently employed commercial techniques as reference tests, measured different immunoglobulin isotypes with a predominance for IgG (>50%) and discriminated between acute and chronic infections. In animals, the most commonly used reference techniques were in-house tests, which almost exclusively detected IgG. Common limitations identified in a large number of studies were some misunderstandings of the terms "gold standard" and "reference test" and the absence of information about the negative and positive control sera used or the exact cutoff employed, which were independent of the quality of the study. There is a lack of analytical validation, with few evaluations of cross-reactivity with other pathogens. Diagnostic odds ratio values showed that indirect ELISA based on native or chimeric antigens performed better than other tests. The reproducibility of serological test results in both humans and animals is not guaranteed due to a lack of relevant information and analytical validation. Thus, several key issues should be considered in the future, including interlaboratory ring trials.