Differential transcriptome study on the damage of testicular tissues caused by chronic infection of T. gondii in mice.

BACKGROUND: Toxoplasma gondii is an intracellular protozoan parasite that is widely distributed in humans and warm-blooded animals. T. gondii chronic infections can cause toxoplasmic encephalopathy, adverse pregnancy, and male reproductive disorders. In male reproduction, the main function of the testis is to provide a stable place for spermatogenesis and immunological protection. The disorders affecting testis tissue encompass abnormalities in the germ cell cycle, spermatogenic retardation, or complete cessation of sperm development. However, the mechanisms of interaction between T. gondii and the reproductive system is unclear. The aims were to study the expression levels of genes related to spermatogenesis, following T. gondii infection, in mouse testicular tissue. METHODS: RNA-seq sequencing was carried out on mouse testicular tissues from mice infected or uninfected with the T. gondii type II Prugniaud (PRU) strain and validated in combination with real-time quantitative PCR and immunofluorescence assays. RESULTS: The results showed that there were 250 significant differentially expressed genes (DEGs) (P < 0.05, |log2fold change| â‰§ 1). Bioinformatics analysis showed that 101 DEGs were annotated to the 1696 gene ontology (GO) term. While there was a higher number of DEGs in the biological process classification as a whole, the GO enrichment revealed a significant presence of DEGs in the cellular component classification. The Arhgap18 and Syne1 genes undergo regulatory changes following T. gondii infection, and both were involved in shaping the cytoskeleton of the blood-testis barrier (BTB). The number of DEGs enriched in the MAPK signaling pathway, the ERK1/2 signaling pathway, and the JNK signaling pathway were significant. The PTGDS gene is located in the Arachidonic acid metabolism pathway, which plays an important role in the formation and maintenance of BTB in the testis. The expression of PTGDS is downregulated subsequent to T. gondii infection, potentially exerting deleterious effects on the integrity of the BTB and the spermatogenic microenvironment within the testes. CONCLUSIONS: Overall, our research provides in-depth insights into how chronic T. gondii infection might affect testicular tissue and potentially impact male fertility. These findings offer a new perspective on the impact of T. gondii infection on the male reproductive system.

A novel DNA vaccine encoding the SRS13 protein administered by electroporation confers protection against chronic toxoplasmosis.

Toxoplasma gondii is an obligate intracellular parasite that can infect a variety of mammals including humans and causes toxoplasmosis. Unfortunately, a protective and safe vaccine against toxoplasmosis hasn't been developed yet. In this study, we developed a DNA vaccine encoding the SRS13 protein and immunized BALB/c mice thrice with pVAX1-SRS13 through the intramuscular route (IM) or intradermally using an electroporation device (ID + EP). The immunogenicity of pVAX1-SRS13 was analyzed by ELISA, Western blot, cytokine ELISA, and flow cytometry. The protective efficacy of the pVAX1-SRS13 was investigated by challenging mice orally with T. gondii PRU strain tissue cysts. The results revealed that pVAX1-SRS13 administered through IM or ID + EP routes induced high level of anti-SRS13 IgG antibody responses (P = 0.0037 and P < 0.0001). The IFN-γ level elicited by the pVAX1-SRS13 (ID + EP) was significantly higher compared to the control group (P = 0.00159). In mice administered with pVAX1-SRS13 (ID + EP), CD8+ cells secreting IFN-γ was significantly higher compared to pVAX1-SRS13 (IM) (P = 0.0035) and the control group (P = 0.0068). Mice vaccinated with the SRS13 DNA vaccine did not induce significant IL-4 level. Moreover, a significant reduction in the number of tissue cysts and the load of T. gondii DNA was detected in brains of mice administered with pVAX1-SRS13 through ID + EP and IM routes compared to controls. In conclusion, the SRS13 DNA vaccine was found to be highly immunogenic and confers strong protection against chronic toxoplasmosis.

MicroRNA mmu-miR-511-5p: A promising Diagnostic Biomarker in Experimental Toxoplasmosis Using Different Strains and Infective Doses in Mice with Different Immune States Before and After Treatment.

PURPOSE: Searching for a novel early diagnostic biomarker for toxoplasmosis, real-time-PCR was currently used to measure the serum mmu-miR-511-5p level in male Swiss-albino mice infected with either; ME49 or RH Toxoplasma gondii (T. gondii) strains. METHODS: Three mice groups were used; (GI) constituted the non-infected control group, while (GII) and (GIII) were experimentally infected with ME49 or RH strains, respectively. GII mice were orally infected using 10 or 20 ME49 cysts (ME-10 and ME-20), both were subdivided into; non-treated (ME-10-NT and ME-20-NT) and were further subdivided into; immunocompetent (ME-10-IC and ME-20-IC) [euthanized 3-days, 1, 2, 6 or 8-weeks post-infection (PI)], and immunosuppressed using two Endoxan® injections (ME-10-IS and ME-20-IS) [euthanized 6- or 8-weeks PI], and spiramycin-treated (ME-10-SP and ME-20-SP) that received daily spiramycin, for one-week before euthanasia. GIII mice individually received 2500 intraperitoneal RH strain tachyzoites, then, were subdivided into; non-treated (RH-NT) [euthanized 3 or 5-days PI], and spiramycin-treated (RH-SP) that were euthanized 5 or 10-days PI (refer to the graphical abstract). RESULTS: Revealed significant upregulation of mmu-miR-511-5p in GII, one-week PI, with gradually increased expression, reaching its maximum 8-weeks PI, especially in ME-20-NT group that received the higher infective dose. Immunosuppression increased the upregulation. Contrarily, treatment caused significant downregulation. GIII recorded significant upregulation 3-days PI, yet, treatment significantly decreased this expression. CONCLUSION: Serum mmu-miR-511-5p is a sensitive biomarker for early diagnosis of ME49 and RH infection (as early as one-week and 3-days, respectively), and its expression varies according to T. gondii infective dose, duration of infection, spiramycin-treatment and host immune status.

Molecular regulation of NLRP3 inflammasome activation during parasitic infection.

Parasitic diseases are a serious global health concern, causing many common and severe infections, including Chagas disease, leishmaniasis, and schistosomiasis. The NLRP3 inflammasome belongs to the NLR (nucleotide-binding domain leucine-rich-repeat-containing proteins) family, which are cytosolic proteins playing key roles in the detection of pathogens. NLRP3 inflammasomes are activated in immune responses to Plasmodium, Leishmania, Toxoplasma gondii, Entamoeba histolytica, Trypanosoma cruzi, and other parasites. The role of NLRP3 is not fully understood, but it is a crucial component of the innate immune response to parasitic infections and its functions as a sensor triggering the inflammatory response to the invasive parasites. However, while this response can limit the parasites' growth, it can also result in potentially catastrophic host pathology. This makes it essential to understand how NLRP3 interacts with parasites to initiate the inflammatory response. Plasmodium hemozoin, Leishmania glycoconjugate lipophosphoglycan (LPG) and E. histolytica Gal/GalNAc lectin can stimulate NLRP3 activation, while the dense granule protein 9 (GRA9) of T. gondii has been shown to suppress it. Several other parasitic products also have diverse effects on NLRP3 activation. Understanding the mechanism of NLRP3 interaction with these products will help to develop advanced therapeutic approaches to treat parasitic diseases. This review summarizes current knowledge of the NLRP3 inflammasome's action on the immune response to parasitic infections and aims to determine the mechanisms through which parasitic molecules either activate or inhibit its action.

Observational longitudinal study on Toxoplasma gondii infection in fattening beef cattle: serology and associated haematological findings.

Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is a globally distributed zoonotic infection with significant implications for human and animal health. This study investigated the prevalence of T. gondii infection in a population of beef cattle at three different stages of their productive lifespan and examined the impact of T. gondii serological status on blood parameters. A commercial beef fattening unit in Italy was the setting for this research, which involved a biosecurity assessment upon cattle arrival, blood sampling at three time points and Toxoplasma-specific serological testing using indirect fluorescent antibody tests (IFAT). Results revealed a dynamic pattern of T. gondii seropositivity in cattle, with an initial prevalence of 30.6% at arrival (T0) that increased to 44.6% at 14 days (T1) and then decreased slightly to 39.3% at slaughter after 5 months (T2). Interestingly, seroconversion was observed during the study, indicating ongoing infections, and antibody waning occurred in some animals. In terms of blood parameters, seropositive cattle exhibited significantly lower mean corpuscular volume (MCV) and a higher neutrophil-lymphocyte (N/L) ratio, suggesting an activation of the innate immune response. Furthermore, cattle with higher antibody titres displayed higher neutrophil counts. However, all blood parameters with a statistical significance were within the reference range. This study provides for the first time a longitudinal investigation on the serological status for T. gondii in naturally exposed beef cattle. These findings provide valuable insights into the clinico-pathological aspects of natural T. gondii exposure in cattle and underscore the importance of monitoring and managing T. gondii infection in livestock production systems.

First Description of Marinoquinoline Derivatives' Activity against Toxoplasma gondii.

Toxoplasmosis is a globally prevalent zoonotic disease with significant clinical implications, including neurotoxoplasmosis, a leading cause of cerebral lesions in AIDS patients. The current pharmacological treatments for toxoplasmosis face clinical limitations, necessitating the urgent development of new therapeutics. Natural sources have yielded diverse bioactive compounds, serving as the foundation for clinically used derivatives. The exploration of marine bacteria-derived natural products has led to marinoquinolines, which feature a pyrroloquinoline core and demonstrate in vitro and in vivo anti-Plasmodium activity. This study investigates the in vitro anti-Toxoplasma gondii potential of six marinoquinoline derivatives. Additionally, it conducts absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions, and evaluates the in vivo efficacy of one selected compound. The compounds displayed half-maximal effective concentration (EC50) values between 1.31 and 3.78 µM and half-maximal cytotoxic concentration (CC50) values ranging from 4.16 to 30.51 µM, resulting in selectivity indices (SI) from 3.18 to 20.85. MQ-1 exhibiting the highest in vitro SI, significantly reduced tachyzoite numbers in the peritoneum of RH-infected Swiss mice when it was orally administered at 12.5 mg/kg/day for eight consecutive days. Also, MQ-1 significantly reduced the cerebral parasite burden in chronically ME49 infected C57BL/6 mice when it was orally administered at 25 mg/kg/day for 10 consecutive days. These findings underscore the promising anti-T. gondii activity of marinoquinolines and their potential as novel therapeutic agents against this disease.

Prevalence of Toxoplasma gondii IgM and IgG positive cats in Los Angeles County, California.

Objectives: The objective of this study was to determine the prevalence of Toxoplasma gondii IgM and IgG positive cats in Los Angeles County, California. T gondii antibodies are common in sera from cats in most reported studies around the world. Although the majority of infected cats never develop clinical disease, development of acute infection and recrudescence of latent infection secondary to immunosuppression has been reported. Knowledge of the serologic status of T gondii may be important when considering immunosuppressive treatments. Methods: T gondii IgM and IgG antibody titers were measured in 225 cats. Sera from owned cats tested at a multispecialty veterinary hospital were included both retrospectively and prospectively (n = 125). Sera from feral cats tested through a collaborating humane society were included prospectively (n = 100). Results: Of the 13 (5.8%) cats with IgM titers, 10 were positive at the minimal cut-off titer (1:64), one cat was clinically ill and none were currently positive for IgG antibodies, suggesting false-positive results for nine cats, giving an adjusted IgM prevalence rate of 1.8% (95% CI 0.7-4.5). A total of five (2.2%) cats were positive for IgG antibodies and no cat was positive for both antibodies. Conclusions and relevance: Most studies of T gondii antibodies in cat sera from California have shown higher prevalence rates, suggesting the cats in this municipality have a low risk of exposure. The study emphasizes that serological test results do not necessarily correlate to the presence of clinical illness.

Application of gold immunochromatographic assay strip combined with digital evaluation for early detection of Toxoplasma gondii infection in multiple species.

BACKGROUND: Timely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results. METHODS: Colloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera. RESULTS: AMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%-23.4% of these samples were found to be positive for T. gondii. CONCLUSIONS: This study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.

Protective effect of arctiin against Toxoplasma gondii HSP70-induced allergic acute liver injury by disrupting the TLR4-mediated activation of cytosolic phospholipase A2 and platelet-activating factor.

Toxoplasma gondii (T. gondii)-derived heat shock protein 70 (T.g.HSP70) is a toxic protein that downregulates host defense responses against T. gondii infection. T.g.HSP70 was proven to induce fatal anaphylaxis in T. gondii infected mice through cytosolic phospholipase A2 (cPLA2) activated-platelet-activating factor (PAF) production via Toll-like receptor 4 (TLR4)-mediated signaling. In this study, we investigated the effect of arctiin (ARC; a major lignan compound of Fructus arctii) on allergic liver injury using T.g.HSP70-stimulated murine liver cell line (NCTC 1469) and a mouse model of T. gondii infection. Localized surface plasmon resonance, ELISA, western blotting, co-immunoprecipitation, and immunofluorescence were used to investigate the underlying mechanisms of action of ARC on T. gondii-induced allergic acute liver injury. The results showed that ARC suppressed the T.g.HSP70-induced allergic liver injury in a dose-dependent manner. ARC could directly bind to T.g.HSP70 or TLR4, interfering with the interaction between these two factors, and inhibiting activation of the TLR4/mitogen-activated protein kinase/nuclear factor-kappa B signaling, thereby inhibiting the overproduction of cPLA2, PAF, and interferon-γ. This result suggested that ARC ameliorates T.g.HSP70-induced allergic acute liver injury by disrupting the TLR4-mediated activation of inflammatory mediators, providing a theoretical basis for ARC therapy to improve T.g.HSP70-induced allergic liver injury.

Analysis of Serum Immunoglobulin G (IgG) Levels against Toxoplasma Gondii, Herpes Simplex Virus Types 1 and 2, Cytomegalovirus and Hepatitis C Virus in Vitiligo.

Background: Pathogen infections have been associated with autoimmunity, which in turn has been implicated in the pathogenesis of vitiligo. However, the association between pathogen infections and vitiligo remains elusive. Aims: This study aimed to assess the proportion of individuals who tested positive for specific IgG antibodies against selected pathogens in patients with vitiligo and control subjects. Materials and Methods: Plasma from 51 patients with vitiligo and 51 age- and gender-matched controls were tested for anti-Toxoplasma gondii (T. gondii) IgG, anti-herpes simplex types 1 and 2 (HSV-1/2) IgG, anti-cytomegalovirus (CMV) IgG and anti-hepatitis C virus IgG. Results: Among all participants (n = 102), 63%, 84% and 87% tested positive for anti-T. gondii, anti-HSV-1/2 and anti-CMV IgG antibodies, respectively. Anti-hepatitis C virus IgG was negative in all samples tested. Positive anti-T. gondii IgG was detected in plasma samples of 39 (78%) patients with vitiligo and 25 (49%) controls (odds ratio [OR] 3.68, 95% confidence interval [CI] 1.55-8.76, P = 0.0036). Anti-HSV-1/2 IgG was detected in samples of 47 (92%) patients with vitiligo and 38 (76%) controls (OR 3.71, 95% CI 1.11-12.44, P = 0.031). Differences in frequencies of positive results for anti-T. gondii IgG and anti-HSV-1/2 IgG were only significant in samples from female patients with vitiligo when compared with controls (P = 0.036 and 0.024, respectively). Anti-CMV IgG was detected in samples from 46 patients with vitiligo (90%) and 41 (84%) controls (P = 0.384). Conclusions: T. gondii IgG and HSV-1/2 IgG were significantly more frequent in patients with vitiligo, especially in women, when compared with age- and gender-matched controls. Since T. gondii and HSV-1/2 infections can trigger autoimmune events, past exposure to these pathogens may be a risk factor for the development of vitiligo.

The Modulated Role of Toxoplasma gondii on Eosinophils in Psychiatric Disorders after Cannabis Cessation.

The aim of our study was to evaluate the impact of T. gondii status on eosinophils count (EOS), the eosinophil-to-lymphocyte ratio (ELR), and the eosinophil-to-neutrophil-to-lymphocytes ratio (ENLR) before and after cannabis cessation in patients with psychiatric disorders. One hundred and eighty-eight patients were included in the study. T. gondii, EOS, ELR, ENLR, and urinary cannabis were measured at baseline and after 4 weeks of cannabis cessation. Highest levels and increase of PNE (p = 0.02), ENLR levels (p = 0.031) and highest level of ELR (p = 0.03) were found in patients after cannabis cessation only in patients positive for T. gondii serology (Toxo+ group). At four weeks, significant interactions between cannabis and T. gondii status for EOS (p = 0.038), and for ENLR (p = 0.043) levels were found, as well as for the evolution between baseline and 4 weeks for ENLR level (p = 0.049). After cannabis cessation, we found a positive correlation between negative symptoms and EOS levels at 4 weeks in the Toxo+ group. This study shows that the increase of inflammation after cannabis cessation might be modulated by T. gondii seropositivity status in patients after cannabis cessation.

Seroprevalence and risk factors of Toxoplasma gondii infection in slaughtered chickens in Tripoli, Lebanon.

Toxoplasma gondii is a cosmopolitan protozoan parasite that has a wide range of intermediate hosts. It infects all warm-blooded animals, including humans and birds. The latter typically pick up the infection by ground feeding, and people can contract the parasite from eating undercooked chicken meat. In recent years, investigations into T. gondii infection in poultry have been reported worldwide. However, there is no epidemiological data regarding the seroprevalence of anti-T. gondii antibodies in chicken in Lebanon. Thus, the current investigation was carried out to determine the seroprevalence and associated risk factors of T. gondii infection in chicken destined for human consumption in the Tripoli district of Lebanon. For this, a cross-sectional study was carried out between April 2021 and February 2022. Blood samples were collected from 400 chickens in four poultry abattoirs in Tripoli. The modified agglutination test (MAT) was used to test sera for T. gondii antibodies. The association of T. gondii seroprevalence with potential risk factors was assessed using the Chi-square test. Multivariate analysis was used to confirm the association. The seroprevalence of T. gondii antibodies reported in this study was 13% (52/400); it was higher in the free-range chicken group (19.3%, 29/150) than in the caged group (9.2%, 23/250) (OR = 2.365; 95% CI: 1.311-4.267) (P = 0.004). The wet season and the presence of cats in the poultry farms were significantly associated with an increased seropositivity to T. gondii infection (P ≤ 0.0001). Given the occurrence of T. gondii antibodies in slaughtered chicken in this area, the consumption of raw or undercooked chicken meats may pose a serious threat to public health and highlight the need to implement appropriate precautionary strategies to halt the spread of T. gondii to humans.

Latent toxoplasmosis in COVID-19 patients and link with higher mortality in COVID-19 male patients.

BACKGROUND: Immunocompromised patients may be at risk for reactivating the toxoplasmosis infection; therefore, early diagnosis would be highly desirable in these individuals. This study evaluated the possible association between coronavirus disease 2019 (COVID-19) and latent Toxoplasma gondii infection in Guilan province, Iran. MATERIALS AND METHODS: The study was performed among 210 COVID-19 patients referred to Guilan University of Medical Sciences hospitals in 2022. Peripheral blood samples were taken for serum separation, collected into tubes, and kept at - 20 °C until use. Blood samples were obtained from COVID-19 patients. IgG antibody to Toxoplasma gondii was detected by a commercial ELISA kit. Accordingly, IgG absorbance levels <9 were considered harmful, 9-11 was considered borderline, and >11 was positive. RESULTS: Toxoplasma IgG antibodies were found in 73.9 % of patients with COVID-19 in male patients. The seroprevalence of Toxoplasma in dead and lived COVID-19 male patients was 83.3 % and 66.7 %, respectively, and this difference was significant. A present study found a significant correlation between the rising titer of Toxoplasma IgG and the severity of COVID-19. There was no significant difference between the hospitalization duration factor and the seropositivity rate. CONCLUSION: Regarding the significant association between the rising titer of Toxoplasma IgG and the severity of COVID-19. The findings demonstrated an association between the severity and mortality rate of COVID-19 with higher titer Anti-Toxoplasma IgG antibodies. Toxoplasmosis is currently considered a risk factor for COVID-19.

Synthesis, X-ray Structure, Hirshfeld, DFT Conformational, Cytotoxic, and Anti-Toxoplasma Studies of New Indole-Hydrazone Derivatives.

The hydrazones 3a-c, were synthesized from the reaction of indole-3-carbaldehyde and nicotinic acid hydrazide, isonicotinic acid hydrazide, and benzoic acid hydrazide, respectively. Their structures were confirmed using FTIR, 1HNMR, and 13CNMR spectroscopic techniques. Exclusively, hydrazones 3b and 3c were confirmed using single crystal X-ray crystallography to exist in the Eanti form. With the aid of DFT calculations, the most stable configuration of the hydrazones 3a-c in gas phase and in nonpolar solvents (CCl4 and cyclohexane) is the ESyn form. Interestingly, the DFT calculations indicated the extrastability of the EAnti in polar aprotic (DMSO) and polar protic (ethanol) solvents. Hirshfeld topology analysis revealed the importance of the N…H, O…H, H…C, and π…π intermolecular interactions in the molecular packing of the studied systems. Distribution of the atomic charges for the hydrazones 3a-c was presented. The hydrazones 3a-c showed a polar character where 3b has the highest polarity of 5.7234 Debye compared to the 3a (4.0533 Debye) and 3c (5.3099 Debye). Regarding the anti-toxoplasma activity, all the detected results verified that 3c had a powerful activity against chronic toxoplasma infection. Compound 3c showed a considerable significant reduction percent of cyst burden in brain homogenates of toxoplasma infected mice representing 49%.

Retinal ferroptosis as a critical mechanism for the induction of retinochoroiditis during ocular toxoplasmosis.

Toxoplasmosis is a major infectious disease, affecting approximately one-third of the world's population; its main clinical manifestation, ocular toxoplasmosis (OT), is a severe sight-threatening disease. Nevertheless, the diagnosis of OT is based on clinical findings, which needs improvement, even with biochemical tests, such as polymerase chain reaction and antibody detections. Furthermore, the efficacy of OT-targeted treatment is limited; thus, additional measures for diagnosis and treatments are needed. Here, we for the first time report a significantly reduced iron concentration in the vitreous humor (VH) of human patients infected with OT. To obtain further insights into molecular mechanisms, we established a mouse model of T. gondii infection, in which intravitreally injected tracer 57Fe, was accumulated in the neurosensory retina. T. gondii-infected eyes showed increased lipid peroxidation, reduction of glutathione peroxidase-4 expression and mitochondrial deformity in the photoreceptor as cristae loss. These findings strongly suggest the involvement of ferroptotic process in the photoreceptor of OT. In addition, deferiprone, an FDA-approved iron chelator, reduced the iron uptake but also ameliorated toxoplasma-induced retinochoroiditis by reducing retinal inflammation. In conclusion, the iron levels in the VH could serve as diagnostic markers and iron chelators as potential treatments for OT.

CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood.

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

The effects of intracellular iron availability on the outcome of Toxoplasma gondii infection in mice.

Toxoplasma gondii (T. gondii) is a parasite that obtains the iron it needs for its own metabolism from the host-cell iron pool. In this work, we aimed to investigate if iron supplementation or deficiency affected the course of T. gondii infection. Eighty mice were divided into four groups, each with 20 animals: Group (I): Uninfected control group. Group (II): Infected control group: injected with Phosphate buffered saline. Group (III): Infected group: received iron sucrose treatment. Group (IV): Infected group: treated with deferoxamine. Quantitative PCR studies were performed on days 3 and 8 post-infection to detect the expression of iron metabolism genes (hamp and ferroprotin) and immune-histochemical analysis to study the percentage of TNF-α and TGF-ß tissue expression. Iron supplementation induced progressions of infection evident by increased tissue expression of pro-inflammatory cytokine TNF-α and downregulation of TGF-ß which is mostly linked to suppression of the inflammatory process caused by T. gondii. Increased expression of TGF-ß and decreased expression of TNF-α was noticed when iron deprivation occurred. On day 3, we noticed increased expression in the hamp gene with iron supplementation while it decreases when the iron supply is low. On the contrary, iron deficiency increased ferroprotin gene expression whereas supplementing decreased it. On day 8, the level of expression of these genes returned to normal levels. These observations document the potential role of iron in controlling toxoplasmosis infection and indicate that the transcription of hamp and ferroprotin in T. gondii-infected cells appears to be regulated by a sophisticated indirect mechanism.

Possible association between Toxoplasma gondii infection and sleep disorders: A systematic review.

BACKGROUND: Few studies investigated the relationship between toxoplasmosis and sleep disorders. Sleep disorders can lead to suicidal behavior and traffic accidents. Therefore, the purpose of this study is to collect information and investigate the possible relationship between Toxoplasma gondii (T. gondii) and sleep disorders. METHODS: To achieve the goal of the present study, five English databases (PubMed, ScienceDirect, Web of Science, Scopus, and ProQuest) were systematically searched for related studies from their inception until October 18, 2022. The obtained articles were screened based on the title, abstract and full text. Then, the quality of the papers investigating the relationship between toxoplasmosis and sleep disorders was evaluated, and finally, the data from the relevant studies were extracted in a Microsoft Excel data sheet. RESULTS: Eight articles (4 case-control and 4 cross-sectional studies) were entered in this systematic review containing 926 patients with sleep disorders and 1877 people without sleep disorders in casecontrol studies, out of which 212 (22.89%) and 392 (20.88%) individuals were positive for anti-T. gondii IgG antibody using different serological methods. Also, 2885 people with sleep disorders were investigated for anti-T. gondii IgG antibody in cross-sectional studies, out of which 1559 (54.03%) cases were positive. CONCLUSION: The results of this study suggest that T. gondii infection may be a risk factor for sleep disorders. However, the number of related studies is small, and there are contradictions in the findings of these articles. Therefore, further studies are necessary to clarify the possible association between T. gondii infections and sleep disorders.

Influenza A Virus Weakens the Immune Response of Mice to Toxoplasma gondii, Thereby Aggravating T. gondii Infection.

This study aimed to investigate the relationship between the T. gondii type II strain (Pru) and respiratory viral infections, specifically focusing on the co-infection with PR8 (influenza A/Puerto Rico/8/34). In this study, we found that the number of T. gondii (Pru) in the lungs of co-infected mice was significantly higher and lesions were more severe than those in the group infected with T. gondii (Pru) alone, whereas IAV (influenza A virus) copy numbers of co-infected and PR8 alone infected groups were negligible, suggesting that infection with IAV increased the pathogenicity of T. gondii (Pru) in mice. The invasion and proliferation assays demonstrated no significant effect of co-infection on T. gondii (Pru) infection or replication in vitro. To further explore the factors causing the altered pathogenicity of T. gondii (Pru) caused by co-infection, we found that decreased expression levels of IL-1ß, IL-6, and IL-12 in the co-infected group were associated with the early immune responses against T. gondii (Pru), which affected the division of T. gondii (Pru). Moreover, the significant decrease in the CD4+/CD8+ ratio indicated a weakened long-term immune killing ability of the host against T. gondii (Pru) following IAV infection. In conclusion, a T. gondii type II strain (Pru) could not be properly cleared by the host immune system after IAV infection, resulting in toxoplasmosis and even death in mice.

Pantothenate biosynthesis in Toxoplasma gondii tachyzoites is not a drug target.

Toxoplasma gondii is a pervasive apicomplexan parasite that can cause severe disease and death in immunocompromised individuals and the developing foetus. The treatment of toxoplasmosis often leads to serious side effects and novel drugs and drug targets are therefore actively sought. In 2014, Mageed and colleagues suggested that the T. gondii pantothenate synthetase, the enzyme responsible for the synthesis of the vitamin B5 (pantothenate), the precursor of the important cofactor, coenzyme A, is a good drug target. Their conclusion was based on the ability of potent inhibitors of the M. tuberculosis pantothenate synthetase to inhibit the proliferation of T. gondii tachyzoites. They also reported that the inhibitory effect of the compounds could be antagonised by supplementing the medium with pantothenate, supporting their conclusion that the compounds were acting on the intended target. Contrary to these observations, we find that compound SW314, one of the compounds used in the Mageed et al. study and previously shown to be active against M. tuberculosis pantothenate synthetase in vitro, is inactive against the T. gondii pantothenate synthetase and does not inhibit tachyzoite proliferation, despite gaining access into the parasite in situ. Furthermore, we validate the recent observation that the pantothenate synthetase gene in T. gondii can be disrupted without detrimental effect to the survival of the tachyzoite-stage parasite in the presence or absence of extracellular pantothenate. We conclude that the T. gondii pantothenate synthetase is not essential during the tachyzoite stage of the parasite and it is therefore not a target for drug discovery against T. gondii tachyzoites.