Toxoplasma Gondii Strains Research Articles

Functional dissection of prenyltransferases reveals roles in endocytosis and secretory vacuolar sorting in type 2-ME49 strain of Toxoplasma gondii

ABSTRACT Prenyltransferases act essential roles in the prenylation modification, which is significant for proteins, like small GTPases to execute various important activities in Toxoplasma gondii (T.gondii). The structures and partial functions of prenyltransferases (FTase, GGTase-I, and GGTase-II) in prenylation process have been dissected in T. gondii. However, the cellular effects of prenyltransferases on type 2-ME49 strain of Toxoplasma are largely unknown. To address this gap, CRISPR/Cas9-based gene-editing technology was employed to construct conditional knockdown strains of prenyltransferases in ME49 strain. Subsequent observation of ingestion ability of host cytosolic molecules (e.g, green fluorescent protein [GFP]) and status of secretory vacuolar sorting post-knockdown of prenyltransferases revealed significant findings. Our study demonstrated that degradation of FTase and GGTase-II notably affected the trafficking of endocytic GFP and vacuolar secretory trafficking to rhoptry bulb. Additionally, depletion of GGTase-II led to disordered endoplasmic reticulum and microtubules, as well as impaired gliding motility. The integrity of mitochondrion was damaged after degradation of GGTase-I. These findings underscore the critical functions of prenyltransferases in endocytosis and secretory vacuolar sorting in ME49 strain of T. gondii, thereby enhancing our understanding of prenyltransferases as potential drug targets.

Enhanced clindamycin delivery using chitosan-coated niosomes to prevent Toxoplasma gondii strain VEG in pregnant mice: an experimental study

BackgroundCongenital toxoplasmosis occurs when a pregnant woman becomes infected with Toxoplasma gondii (T. gondii) for the first time. Treatment typically involves antimicrobial medications, with spiramycin commonly used to prevent transmission. However, spiramycin's effectiveness is limited due to poor placental penetration. Clindamycin, another antibiotic, can cross the placenta but reaches the fetus at only half the maternal concentration. Encapsulating the drug in chitosan-coated niosomes (Cs-Nio) could enhance its effectiveness by targeting specific organs and ensuring sustained release. To address the challenges of using clindamycin, a niosome-coated chitosan system was investigated for treating congenital toxoplasmosis caused by the VEG strain of T. gondii in an animal model.MethodsPregnant mice were infected with VEG strain of T. gondii on the 12th day of pregnancy, followed by treatment with various drugs across six groups. The treatments included chitosan-coated niosomes loaded clindamycin (Cs-Nio-Cli) and other controls. Parasitological evaluations (microscopic examination and real-time PCR), along with histopathological and immunological assessments were conducted to assess treatment efficacy. Finally, statistical analysis was conducted using GraphPad Prism 8.0 and SPSS 26, comparing test and control groups with T test and Mann–Whitney test. A p ≤ 0.05 was considered statistically significant.ResultsThe study found that treatment with Cs-Nio-Cli significantly reduced the number of T. gondii cysts in the brain and eyes (97.59% and 92.68%, respectively) compared to the negative control group. It also mitigated inflammatory changes, prevented cell death, and reduced vascular cuffs in the brain. In addition, Cs-Nio-Cli treatment decreased bleeding, placental thrombosis, and inflammatory cell infiltration in the placenta while improving eye tissue health by reducing retinal folds and bleeds. Immunologically, nanoclindamycin treatment resulted in lower TNF-α cytokine levels and higher IL-10 levels, indicating an enhanced anti-inflammatory response.ConclusionsAlthough Cs-Nio-Cli demonstrates promise in reducing the transmission of congenital toxoplasmosis and mitigating the effects of congenital toxoplasmosis, additional research is necessary to determine the optimal treatment regimens for the complete eradication of the parasite in the fetus.

Live-attenuated PruΔgra72 strain of Toxoplasma gondii induces strong protective immunity against acute and chronic toxoplasmosis in mice

BackgroundToxoplasma gondii is an intracellular opportunistic pathogenic protozoan that poses serious threats, particularly in immunocompromised individuals. In the absence of a robust prophylactic measure, the mitigation and management of toxoplasmosis present formidable challenges to public health. We recently found that GRA72 plays an important role in parasitophorous vacuole (PV) morphology, growth and virulence of T. gondii. However, whether gra72-deficient strain can be used as a vaccine remains unknown.MethodsWe first examined the attenuated virulence of gra72 gene knockout strain (PruΔgra72) and the parasite load in organs of the infected mice. Subsequently, we evaluated the immune-protective effects of the PruΔgra72 vaccination against challenge with various types of T. gondii tachyzoites and Pru cysts. Furthermore, levels of antibodies and cytokines induced by PruΔgra72 vaccination were examined. Statistical analysis was conducted by Student’s t-test or Mantel-Cox log-rank test based on data obtained from three independent experiments with GraphPad Prism 8.0.ResultsWe found that PruΔgra72 strain exhibited a significantly attenuated virulence even at the highest dose of 5 × 107 tachyzoites in Kunming mice model. The significant decrease of brain cyst burden and parasite load in the organs of the PruΔgra72-infected mice suggested its potentiality as a live-attenuated vaccine. Hence, we explored the protective immunity of PruΔgra72 vaccination against toxoplasmosis. Results showed that vaccination with 5 × 106 PruΔgra72 tachyzoites triggered a strong and sustained Th1-biased immune response, marked by significantly increased levels of anti-T. gondii IgG antibodies, and significantly higher levels of Th1 type cytokines (IL-2, IL-12 and IFN-γ) compared to that of Th2 type (IL-4 and IL-10). Vaccination with 5 × 106 PruΔgra72 tachyzoites in mice conferred long-term protection against T. gondii infection by less virulent tachyzoites (ToxoDB#9 PYS and Pru strains) and Pru cysts, provided partial protection against acute infection by high virulent Type I RH tachyzoites and significantly decreased brain cyst burden of chronically infected mice.ConclusionsThe avirulent PruΔgra72 induced strong protective immunity against acute and chronic T. gondii infection and is a promising candidate for developing a safe and effective live-attenuated vaccine against T. gondii infection.Graphical

Functional Characterization of 11 Tentative Microneme Proteins in Type I RH Strain of Toxoplasma gondii Using the CRISPR-Cas9 System.

Toxoplasma gondii, a pathogenic apicomplexan parasite, infects approximately one third of the world's population and poses a serious threat to global public health. Microneme proteins (MICs) secreted by the microneme, an apical secretory organelle of T. gondii, play important roles in the invasion, motility, and intracellular survival of T. gondii. In this study, we selected 11 genes of interest (GOIs) of T. gondii, tentative MICs predicted to be localized in micronemes, and we used the CRISPR-Cas9 system to construct epitope tagging strains and gene knockout strains to explore the localization and function of these 11 tentative MICs. Immunofluorescence assay showed that nine tentative MICs (TGME49_243930, TGME49_200270, TGME49_273320, TGME49_287040, TGME49_261710, TGME49_205680, TGME49_304490, TGME49_245485, and TGME49_224620) were localized or partially localized in the microneme, consistent with the prediction. However, TGME49_272380 and TGME49_243790 showed different localizations from the prediction, being localized in the endoplasmic reticulum and the dense granule, respectively. Further functional characterization of the 11 RHΔGOI strains revealed that deletion of these 11 GOIs had no significant effect on plaque formation, intracellular replication, egress, invasion ability, and virulence of T. gondii. Although these 11 GOIs are not essential genes for the growth and virulence of tachyzoites of type I RH strain, they may have potential roles in other developmental stages or other genotypes of T. gondii. Thus, further research should be performed to explore the possible role of the nine mics and the other two GOIs in other life cycle stages and other genotypes of T. gondii.

Therapeutic potential of Garcinia kola against experimental toxoplasmosis in rats.

Cerebral toxoplasmosis, the most common opportunistic infection in immunocompromised individuals, is increasingly reported in immunocompetent individuals due to mutant strains of Toxoplasma gondii, which, furthermore, are reported to be resistant to available treatments. We assessed the therapeutic potential of Garcinia kola, a medicinal plant reported to have antiplasmodial and neuroprotective properties, against experimental toxoplasmosis in rats. Severe toxoplasmosis was induced in male Wistar rats (156.7 ± 4.1 g) by injecting them with 10 million tachyzoites in suspension in 500 µl of saline (intraperitoneal), and exclusive feeding with a low-protein diet [7% protein (weight by weight)]. Then, animals were treated with hexane, dichloromethane, and ethyl acetate fractions of Garcinia kola. Footprints were analysed and open-field and elevated plus maze ethological tests were performed when symptoms of severe disease were observed in the infected controls. After sacrifice, blood samples were processed for Giemsa staining, organs were processed for haematoxylin and eosin staining, and brains were processed for Nissl staining and cell counting. Compared with non-infected animals, the infected control animals had significantly lower body weights (30.27%↓, P = 0.001), higher body temperatures (P = 0.033) during the sacrifice, together with signs of cognitive impairment and neurologic deficits such as lower open-field arena centre entries (P < 0.001), elevated plus maze open-arm time (P = 0.029) and decreased stride lengths and step widths (P < 0.001), as well as neuronal loss in various brain areas. The ethyl acetate fraction of Garcinia kola prevented or mitigated most of these signs. Our data suggest that the ethyl acetate fraction of Garcinia kola has therapeutic potential against cerebral toxoplasmosis.

Antiparasitic and apoptotic modulatory activities of curcumin and extracts of Nigella sativa L, Zingiber officinale Rosc., and Punica granatum L. in combination with spiramycin against chronic cerebral toxoplasmosis in immunocompromised mice

ObjectiveTo examine the effect of coadministration of different medicinal plant extracts (curcumin and extracts of Nigella sativa L. [N. sativa, Jia Hei Zhong Cao], Zingiber officinale Rosc. [Z. officinale, Sheng Jiang], and Punica granatum L. [P. granatum, Shi Liu]) with spiramycin (SP) against the cystogenic ME-49 Toxoplasma gondii (T. gondii) strain in immunocompromised mice. MethodsWe utilized 68 mice categorized into 8 groups: 2 non-infected controls (immunocompetent and immunocompromised), 1 infected control, and 5 infected and treated groups. Following the experiment, the cerebral tissues of each mouse underwent parasitological, histopathological, and immunohistochemical evaluations. ResultsCompared with the infected non-treated group, all infected treated groups showed significant reductions in brain cyst numbers (all P < .05), with the highest reduction rate (77.4%) recorded for P. granatum and SP combination (G8). Only G8 showed a significant reduction in mouse deaths compared with the other groups. With regards to restoring histopathological changes and decreasing inflammation, the groups infected and treated with curcumin and P. granatum combined with SP showed the best results (P < .05). Combinations of curcumin and extracts of Z. officinale and P. granatum with SP significantly restored the cerebral expression of caspase-3 compared with the N. sativa extract combination (P < .05). G8 showed non-significant expression compared with the local expression in the negative control groups. ConclusionOur study revealed that the coadministration of P. granatum extract with SP was the most effective combination against chronic cerebral toxoplasmosis.

A combination of GRA3, GRA6 and GRA7 peptides offer a useful tool for serotyping type II and III Toxoplasma gondii infections in sheep and pigs.

The clinical consequences of toxoplasmosis are greatly dependent on the Toxoplasma gondii strain causing the infection. To better understand its epidemiology and design appropriate control strategies, it is important to determine the strain present in infected animals. Serotyping methods are based on the detection of antibodies that react against segments of antigenic proteins presenting strain-specific polymorphic variations, offering a cost-effective, sensitive, and non-invasive alternative to genotyping techniques. Herein, we evaluated the applicability of a panel of peptides previously characterized in mice and humans to serotype sheep and pigs. To this end, we used 51 serum samples from experimentally infected ewes (32 type II and 19 type III), 20 sheep samples from naturally infected sheep where the causative strain was genotyped (18 type II and 2 type III), and 40 serum samples from experimentally infected pigs (22 type II and 18 type III). Our ELISA test results showed that a combination of GRA peptide homologous pairs can discriminate infections caused by type II and III strains of T. gondii in sheep and pigs. Namely, the GRA3-I/III-43 vs. GRA3-II-43, GRA6-I/III-213 vs. GRA6-II-214 and GRA6-III-44 vs. GRA6-II-44 ratios showed a statistically significant predominance of the respective strain-type peptide in sheep, while in pigs, in addition to these three peptide pairs, GRA7-II-224 vs. GRA7-III-224 also showed promising results. Notably, the GRA6-44 pair, which was previously deemed inefficient in mice and humans, showed a high prediction capacity, especially in sheep. By contrast, GRA5-38 peptides failed to correctly predict the strain type in most sheep and pig samples, underpinning the notion that individual standardization is needed for each animal species. Finally, we recommend analyzing for each animal at least 2 samples taken at different time points to confirm the obtained results.

Correlations between the degree of infection by wild strain of Toxoplasma gondii in vitro and porcine hematological parameters

The apicomplexa Toxoplasma gondii is capable of actively proliferating in numerous types of nucleated cells, and therefore has a high potential for dissemination and resistance. Thus, the present work aimed to correlate the inoculum concentrations and amount of post-infection parasites with porcine hematological parameters (including biochemistry) through in vitro culture. Porcine blood was incubated with different concentrations of parasites (1.2 × 107, 6/3/1.5 × 106 cells/mL), then the concentrations of red blood cells (RBC) and their morphology, total and differential leukocytes, and free peptides were evaluated. In addition, eight different blood samples analyzed before inoculation, where subsequent multivariate analysis was applied to correlate different variables with trophozoite concentration. The results showed no significant variation (p < 0.05) in the relative levels of free peptides, or the relative percentage of RBC at all the parasite concentrations tested. However, the normalized percentages of leukocytes and neutrophils showed a significant reduction, while those of lymphocytes, eosinophils and monocytes showed the opposite behavior. Semi-automatic processing of images exhibited significant microcytosis and hypochromia. The multivariate analysis revealed a positive correlation between the amount number of protozoa (AP) and the variables: “Red cells” and “Neutrophils”, an indifference between the AP and the content of free peptides, and the concentration of monocytes in the samples; and a negative correlation for AP and the percentages of lymphocytes and eosinophils. Our results suggest that specific changes in hematological parameters may be associated with different degrees of parasitemia, demanding a thorough diagnostic process and adequate treatment.

Iron depletion has different consequences on the growth and survival of Toxoplasma gondii strains

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite responsible for a pathology called toxoplasmosis, which primarily affects immunocompromised individuals and developing foetuses. The parasite can scavenge essential nutrients from its host to support its growth and survival. Among them, iron is one of the most important elements needed to sustain basic cellular functions as it is involved in a number of key metabolic processes, including oxygen transport, redox balance, and electron transport. We evaluated the effects of an iron chelator on the development of several parasite strains and found that they differed in their ability to tolerate iron depletion. The growth of parasites usually associated with a model of acute toxoplasmosis was strongly affected by iron depletion, whereas cystogenic strains were less sensitive as they were able to convert into persisting developmental forms that are associated with the chronic form of the disease. Ultrastructural and biochemical characterization of the impact of iron depletion on parasites also highlighted striking changes in both their metabolism and that of the host, with a marked accumulation of lipid droplets and perturbation of lipid homoeostasis. Overall, our study demonstrates that although acute iron depletion has an important effect on the growth of T. gondii, it has a more profound impact on actively dividing parasites, whereas less metabolically active parasite forms may be able to avoid some of the most detrimental consequences.

Exploring the energetic metabolism of Toxoplasma gondii: evaluating the impact of atovaquone

Toxoplasmosis is a disease with a wide host range and is considered a public health problem that affects approximately one third of the world's population. In adults, it ranges from benign to ocular and neurological infections and may be more severe in immunocompromised individuals and when transmitted congenitally. The toxoplasmosis treatment protocol, which has remained the same since the 1990s, is considered complex as the drugs currently used do not eliminate the parasite. Therefore, this study aims to evaluate the in vitro metabolic impact of atovaquone on acute (RH) and cystogenic (ME49) strains of Toxoplasma gondii. The strains were cultured and exposed to different concentrations of atovaquone for 7 days. Glycolysis, lactic fermentation, and tricarboxylic acid cycle (TCA) metabolites were quantified by high-performance liquid chromatography (HPLC) and spectrophotometry. The replication rate was drastically impaired in both strains when exposed to 100 nM of atovaquone. After exposure to the drug, glucose was significantly reduced in both strains, being below detection levels for the longest time evaluated, and phosphoenolpyruvate (PEP) behaved similarly. Lactic fermentation was impaired in the RH strain, where lactate was found to be reduced in relation to the control group. The TCA cycle was partially affected in both strains due to a significant decrease in oxaloacetate and α-ketoglutarate concentrations throughout the experimental period. The impact of atovaquone on the energy metabolism of T. gondii may have contributed to the decrease in the parasite's replication rate, demonstrating a promising alternative option for the treatment of toxoplasmosis.

Inflammatory response and parasite regulation in acute toxoplasmosis: the role of P2X7 receptor in controlling virulent atypical genotype strain of Toxoplasma gondii.

Toxoplasmosis is a globally significant disease that poses a severe threat to immunocompromised individuals, especially in Brazil, where a high prevalence of virulent and atypical strains of Toxoplasma gondii is observed. In 1998, the EGS strain, exhibiting a unique infection phenotype, was isolated in Brazil, adding to the complexity of strain diversity. The P2X7 receptor is critical in inflammation and controlling intracellular microorganisms such as T. gondii. However, its genetic variability can result in receptor dysfunction, potentially worsening susceptibility. This study investigates the role of the P2X7 receptor during acute infection induced by the EGS atypical strain, offering insight into the mechanisms of T. gondii infection in this context. We infected the female C57BL/6 (WT) or P2X7 knockout (P2X7-/-) by gavage. The EGS infection causes intestinal inflammation. The P2X7-/- mice presented higher parasite load in the intestine, spleen, and liver. The absence of the P2X7 receptor disrupts inflammatory cell balance by reducing NLRP3, IL-1β, and Foxp3 expression while increasing IFN-γ expression and production in the intestine. In the liver, P2X7-/- animals demonstrate diminished inflammatory infiltrate within the portal and lobular regions concurrent with an enlargement of the spleen. In conclusion, the infection of mice with the EGS strain elicited immune alterations, leading to acute inflammation and cytokine dysregulation, while the P2X7 receptor conferred protection against parasitic proliferation across multiple organs.

Morphometric analysis of tachyzoites of the RH strain of Toxoplasma gondii after in vitro exposure to atovaquone

Morphometric analysis of tachyzoites of the RH strain of Toxoplasma gondii after in vitro exposure to atovaquone

Diversity of genes encoding immune-related GTPase B2 protein, an inherited element responsible for resistance against virulent Toxoplasma gondiistrains, among wild Mus musculus in local area of Japan.

The major genetic group of Toxoplasma gondii, known as type I, generally displays high lethality in laboratory Mus musculus (mouse) strains, with few exceptions. However, because rodents are the primary reservoir hosts for T. gondii, if this characteristic manifests in the wild, type I strains would be extinct. Therefore, we hypothesized that populations of wild rodents capable of harboring type I T. gondii asymptomatically exist globally and are not limited to a few localized areas, as previously thought. The strength of mouse resistance to T. gondii is known to depend on the affinity of the mouse-expressed immunity-related GTPases B2 (IRGB2) protein for the T. gondii-expressed rphoptry protein 5B (ROP5B) protein. Therefore, the Irgb2 gene sequences of 12 individuals mice captured at two animal farms in Gifu Prefecture, and on an island in Okinawa Prefecture, Japan were determined, and subjected to a molecular phylogenetic analysis together with those of various mouse strains worldwide. The Irgb2 gene of M. musculus individuals captured on one farm and one island showed diverse sequences. The sequences from two individual mice captured in an animal farm formed a single clade with a wild mouse derived CAST/EiJ strain, known for its exceptional resistance to type I T. gondii lethality. These results suggest that M. musuculus individuals resistant to the Type I T. gondii strain may be present in Japan, in addition to the previously known populations in South Asia, Thailand and India.

The Brazilian Toxoplasma gondii strain BRI caused greater inflammation and impairment in anxiogenic behavior in mice, which was reverted by rosuvastatin treatment.

This study aimed to investigate the effect of rosuvastatin treatment on anxiety-related behavior and short- and long-term memory impairment in mice infected with acute RH and BRI strains of Toxoplasma gondii. Balb/C mice were infected intraperitoneally and after 2h, oral treatment with rosuvastatin (40mg/kg/day) was initiated for 4days. Behaviors related to anxiety and locomotion were evaluated in the open field (OF), and short- and long-term memory through the novel object recognition test (NOR). At the end of the experiments, peritoneal fluid, brain, liver, and lung were collected for T. gondii DNA quantification and histopathological analysis. Infection with BRI strain reduced the dwell time and central locomotion in the OF (p < 0.05), indicating anxiogenic type behavior, while treatment with rosuvastatin reversed this response (p < 0.05). RH strain infection did not alter any behavior in the OF (p > 0.05) and both strains impaired short- and long-term memory (NOR test), but with no significant treatment effect (p > 0.05). The BRI strain was shown to be more damaging in relation to anxiogenic type behavior when compared to the RH strain (p < 0.05), whereas rosuvastatin reduced this damaging effect in BRI. The treatment reduced the parasite load in the peritoneal lavage, liver, and lung of animals infected with both acute strains; however, it significantly (p < 0.05) attenuated the inflammatory process only in BRI-infected and treated animals, showing that non-archetypal genotypes are more damaging in rodents. This suggests that rosuvastatin may be a drug with great therapeutic potential against T. gondii mainly to reduce damage from virulent strains.

Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method

PurposeA new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains.MethodsT. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples.ResultsAmong type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing.ConclusionThe new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Gut commensal bacteria exacerbate toxoplasmosis associated with TgSheepCHn5 (ToxoDB#2) and TgRedpandaCHn1 (ToxoDB#20) through Th1 immune response.

Oral infection of mice with several strains of Toxoplasma gondii results in intestinal pathological lesions, which contributes to the invasion of this parasite. However, the exact mechanism is unclear, and only a few strains have been explored. Here, T. gondii TgSheepCHn5 and TgRedpandaCHn1 strains from sheep and red panda were evaluated. The TgSheepCHn5 and TgRedpandaCHn1 strains induced intestinal lesions, loss of Paneth cells, and gut commensal bacteria dysbiosis in Swiss Webster mice. The lesions and loss of Paneth cells were dependent on IFN-γ and gut commensal bacteria during T. gondii infection. Deleting IFN-γ or gut commensal bacteria suppressed the Th1 immune response, alleviated the lesions and parasite loading, and upregulated the number of Paneth cells. Loss of IFN-γ production accelerated mice death, whereas the deletion of gut commensal bacteria enhanced the survival time of the host. The Th1 cell immune responses have positive and negative effects on toxoplasmosis, resistance to T. gondii infection, and acceleration intestine lesions. Adjustment of Th1 cell responses and gut commensal bacteria may be effective treatments for toxoplasmosis.

Fatal toxoplasmosis in a captive squirrel monkey (Saimiri boliviensis) in Portugal

New World monkeys are especially vulnerable to develop severe clinical manifestations and succumb to acute toxoplasmosis. This study aimed to describe the histopathological findings and genotypic characterization of the Toxoplasma gondii strain involved in a lethal case occurring in a zoo-housed black-capped squirrel monkey (Saimiri boliviensis) in Portugal. Cyst-like structures suggestive of Sarcocystidae parasites and acute injuries in liver and brain were observed by light microscopy examination. By immunohistochemistry, calprotectin, T. gondii antigen and Iba1 antigen had a positive signaling in lung, liver and brain tissues. Toxoplasma gondii B1, ITS1 and 529 repetitive element fragments amplifications together with the genotyping of 13 microsatellite markers confirmed a systemic T. gondii infection linked to a non-clonal type II strain. This description is consistent to the majority T. gondii strains circulating in Europe.

Chronic infection by atypical Toxoplasma gondii strain induces disturbance in microglia population and altered behaviour in mice

Toxoplasma gondii chronic infection is characterized by the establishment of tissue cysts in the brain and increased levels of IFN-γ, which can lead to brain circuitry interference and consequently abnormal behaviour in mice. In this sense, the study presented here sought to investigate the impact of chronic infection by two T. gondii strains in the brain of infection-resistant mice, as a model for studying the involvement of chronic neuroinflammation with the development of behavioural alterations. For that, male BALB/c mice were divided into three groups: non-infected (Ni), infected with T. gondii ME49 clonal strain (ME49), and infected with TgCkBrRN2 atypical strain (CK2). Mice were monitored for 60 days to establish the chronic infection and then submitted to behavioural assessment. The enzyme-linked immunosorbent assay was used for measurement of specific IgG in the blood and levels of inflammatory cytokines and neurotrophic factors in the brain, and the cell's immunophenotype was determined by multiparametric flow cytometry. Mice infected with ME49 clonal strain displayed hyperlocomotor activity and memory deficit, although no signs of depressive- and/or anxiety-like behaviour were detected; on the other hand, chronic infection with CK2 atypical strain induced anxiety- and depressive-like behaviour. During chronic infection by CK2 atypical strain, mice displayed a higher number of T. gondii brain tissue cysts and inflammatory infiltrate, composed mainly of CD3+ T lymphocytes and Ly6Chi inflammatory monocytes, compared to mice infected with the ME49 clonal strain. Infected mice presented a marked decrease of microglia population compared to non-infected group. Chronic infection with CK2 strain produced elevated levels of IFN-γ and TNF-ɑ in the brain, decreased NGF levels in the prefrontal cortex and striatum, and altered levels of fractalkine (CX3CL1) in the prefrontal cortex and hippocampus. The persistent inflammation and the disturbance in the cerebral homeostasis may contribute to altered behaviour in mice, as the levels of IFN-γ were shown to be correlated with the behavioural parameters assessed here. Considering the high incidence and life-long persistence of T. gondii infection, this approach can be considered a suitable model for studying the impact of chronic infections in the brain and how it impacts in behavioural responses.

Evaluation of the protective immune response of an attenuated strain of Toxoplasma gondii with long-term passages on the Gecko cell line

Toxoplasma gondii is an obligate intracellular parasite that causes the zoonoses disease, named toxoplasmosis, with global prevalence. Until now, no cost-effective treatment method has been found to deal with toxoplasma, and vaccination is the best way to deal with the infection. In the case of pathogenic protozoa, mainly live vaccines have had successful results compared to other vaccine platforms. This study evaluated the efficacy of a live experimental vaccine through long-term passages on the Gecko cell line (Z1) in inducing a protective immune response in BALB/c mice. Thirty mice were divided into three equal groups; G1: the immunized/challenged group (injection of attenuated strain), G2: the immunized/unchallenged group (injection of attenuated strain), and G3: the control group (injection of culture medium).One month after immunization, the studied mice were challenged with 1ₓ103 live tachyzoites of Toxoplasma acute RH strain. We performed Serological investigations, including evaluating antibodies, interferon-gamma (IFN-γ), and interleukins 2, 4, 10, and 12 (IL-2,4,10,12). At the study's end, a molecular test was performed on brain and liver tissues in the immunized groups to check the presence of parasites. The results from the serological tests for the evaluation of antibodies, interferon-gamma (IFN-γ), and interleukins 10 and 12 (IL-10, 12) show a significant difference (p < 0.05) between the vaccinated group and the control group, which are essential indicators of protective immunity against toxoplasma infection. Thus, in the vaccinated group, the survival rate of mice against the challenge was 70%. Also, in group two (G2), the attenuated strain of Toxoplasma gondii had no pathogenicity, and all mice survived until the end of the study period. Molecular results also showed the absence of parasites in the brain and liver tissues in this immunized group and the parasite was found in only one case of liver tissue in G1. Therefore, the attenuated strain has caused significant and protective humoral and cellular immune responses in vaccinated groups. This study showed that with the long-term passage of the acute strain on the Gecko cell line, it is possible to quickly obtain a non-diseased attenuated strain with the ability to induce protective immunity. This successful finding can introduce further research to achieve a promising vaccine in the target animals.

Variation in CD8 T cell IFNγ differentiation to strains of Toxoplasma gondii is characterized by small effect QTLs with contribution from ROP16.

Toxoplasma gondii induces a strong CD8 T cell response characterized by the secretion of IFNγ that promotes host survival during infection. The initiation of CD8 T cell IFNγ responses in vitro differs widely between clonal lineage strains of T. gondii, in which type I strains are low inducers, while types II and III strains are high inducers. We hypothesized this phenotype is due to a polymorphic "Regulator Of CD8 T cell Response" (ROCTR). Therefore, we screened F1 progeny from genetic crosses between the clonal lineage strains to identify ROCTR. Naïve antigen-specific CD8 T cells (T57) isolated from transnuclear mice, which are specific for the endogenous and vacuolar TGD057 antigen, were measured for their ability to become activated, transcribe Ifng and produce IFNγ in response to T. gondii infected macrophages. Genetic mapping returned four non-interacting quantitative trait loci (QTL) with small effect on T. gondii chromosomes (chr) VIIb-VIII, X and XII. These loci encompass multiple gene candidates highlighted by ROP16 (chrVIIb-VIII), GRA35 (chrX), TgNSM (chrX), and a pair of uncharacterized NTPases (chrXII), whose locus we report to be significantly truncated in the type I RH background. Although none of the chromosome X and XII candidates bore evidence for regulating CD8 T cell IFNγ responses, type I variants of ROP16 lowered Ifng transcription early after T cell activation. During our search for ROCTR, we also noted the parasitophorous vacuole membrane (PVM) targeting factor for dense granules (GRAs), GRA43, repressed the response suggesting PVM-associated GRAs are important for CD8 T cell activation. Furthermore, RIPK3 expression in macrophages was an absolute requirement for CD8 T cell IFNγ differentiation implicating the necroptosis pathway in T cell immunity to T. gondii. Collectively, our data suggest that while CD8 T cell IFNγ production to T. gondii strains vary dramatically, it is not controlled by a single polymorphism with strong effect. However, early in the differentiation process, polymorphisms in ROP16 can regulate commitment of responding CD8 T cells to IFNγ production which may have bearing on immunity to T. gondii.