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Infections by the protozoan parasite Toxoplasma gondii are widely prevalent in humans and animals in Brazil. The burden of clinical toxoplasmosis in humans is considered to be very high. The high prevalence and encouragement of the Brazilian Government provides a unique opportunity for international groups to study the epidemiology and control of toxoplasmosis in Brazil. Many early papers on toxoplasmosis in Brazil were published in Portuguese and often not available to scientists in English-speaking countries. In the present paper we review prevalence, clinical spectrum, molecular epidemiology, and control of T. gondii in humans and animals in Brazil. This knowledge should be useful to biologists, public health workers, veterinarians, and physicians. Brazil has a very high rate of T. gondii infection in humans. Up to 50% of elementary school children and 50–80% of women of child-bearing age have antibodies to T. gondii. The risks for uninfected women to acquire toxoplasmosis during pregnancy and fetal transmission are high because the environment is highly contaminated with oocysts. The burden of toxoplasmosis in congenitally infected children is also very high. From limited data on screening of infants for T. gondii IgM at birth, 5–23 children are born infected per 10 000 live births in Brazil. Based on an estimate of 1 infected child per 1000 births, 2649 children with congenital toxoplasmosis are likely to be born annually in Brazil. Most of these infected children are likely to develop symptoms or signs of clinical toxoplasmosis. Among the congenitally infected children whose clinical data are described in this review, several died soon after birth, 35% had neurological disease including hydrocephalus, microcephaly and mental retardation, 80% had ocular lesions, and in one report 40% of children had hearing loss. The severity of clinical toxoplasmosis in Brazilian children may be associated with the genetic characteristics of T. gondii isolates prevailing in animals and humans in Brazil.

Toxoplasma gondii , an obligatory intracellular parasite, is the causative agent of toxoplasmosis, a widespread disease affecting approximately one-third of the global population. This study investigates the strain-specific effects of T. gondii infection on immune responses, reproductive physiology, and oxidative stress in Wistar rats, comparing the highly virulent RH strain to the less virulent VEG strain. The results show that the RH strain significantly reduced levels of the anti-inflammatory cytokine IL-10 ( p  < 0.01) while increasing pro-inflammatory IFN-γ ( p  < 0.05), suggesting a strong inflammatory response. In contrast, the VEG strain produced a more balanced immune profile, with no significant change in IL-10 and a moderate rise in IFN-γ. Although no visible damage to ovarian tissue was observed in any group, the RH strain resulted in a higher number of growing follicles ( p  < 0.05), while the VEG strain led to significantly larger follicles ( p  < 0.05). Both strains elevated CRP levels, with the RH strain inducing a more significant inflammatory response. However, oxidative stress markers showed no significant differences among the experimental groups. In conclusion, the findings indicate that the highly virulent RH strain elicits a strong inflammatory response, whereas the less virulent VEG strain induces a more moderate immune reaction, without causing significant damage to ovarian tissue.

Toxoplasma gondii (T. gondii) ranks as the third most common parasitic parasite worldwide, and it is estimated that > 60% of the population is infected with T. gondii worldwide at some point in their lives. So. Therefore, it is highly curious that T. gondii could be a potential cause of idiopathic infertility that is incidental to male partners, who are responsible for nearly 50% of cases. Testicular histomorphometric analysis was developed to investigate fertility problems. The objective of this experimental study was to assess the impact of toxoplasmosis on spermatogenesis indicated by histomorphometric changes in rat testes and its correlation with the degree of infection in the brain. Ninety male Wistar albino rats were infected with T. gondii, and 30 male rats composed the control group. The studied parameters were investigated from the seventh week until the twelfth week postinfection by estimating the body weight, the weight of the testes, histopathological examination, and metric analysis of the testes. Each time, correlations were detected between the investigated parameters and the infection severity calculated by estimating the cyst burden in brain homogenates and brain lesion grading of stained histological sections.Our findings demonstrated a significant adverse impact of toxoplasmosis on absolute body weight, testis weight, and testis histomorphometry. The grading of brain lesions and the number of brain cysts paralleled each other. There was a reverse correlation between the gonado-somatic index and the number of brain cysts and brain lesion grade. There was a statistically significant correlation between the brain cyst count and the brain lesion grade and the indices 20 A, 20b, 250, and 200 of the testes metric analysis. Conclusion: Our results revealed that toxoplasmosis has an adverse impact on male rat spermatogenic cells, which in turn affects spermatogenesis and fertility. This impact is significantly correlated with the degree of latent infection in the brain.

Toxoplasma gondii ( T. gondii ) is a protozoan parasite that infects approximately one-third of the global human population, often leading to chronic infection. While acute T. gondii infection can cause neural damage in the central nervous system and result in toxoplasmic encephalitis, the consequences of T. gondii chronic infection (TCI) are generally asymptomatic. However, emerging evidence suggests that TCI may be linked to behavioral changes or mental disorders in hosts. Astrocyte polarization, particularly the A1 subtype associated with neuronal apoptosis, has been identified in various neurodegenerative diseases. Nevertheless, the role of astrocyte polarization in TCI still needs to be better understood. This study aimed to establish a mouse model of chronic TCI and examine the transcription and expression levels of glial fibrillary acidic protein (GFAP), C3, C1q, IL-1α, and TNF-α in the brain tissues of the mice. Quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay, and Western blotting were employed to assess these levels. Additionally, the expression level of the A1 astrocyte-specific marker C3 was evaluated using indirect fluorescent assay (IFA). In mice with TCI, the transcriptional and expression levels of the inflammatory factors C1q, IL-1α, and TNF-α followed an up-down-up pattern, although they remained elevated compared to the control group. These findings suggest a potential association between astrocyte polarization towards the A1 subtype and synchronized changes in these three inflammatory mediators. Furthermore, immunofluorescence assay (IFA) revealed a significant increase in the A1 astrocytes (GFAP + C3 + ) proportion in TCI mice. This study provides evidence that TCI can induce astrocyte polarization, a biological process that may be influenced by changes in the levels of three inflammatory factors: C1q, IL-1α, and TNF-α. Additionally, the release of neurotoxic substances by A1 astrocytes may be associated with the development of TCI.

Omega-3 polyunsaturated fatty acids (ω3-PUFAs) have potential protective activity in a variety of infectious diseases, but their actions and underlying mechanisms in Toxoplasma gondii infection remain poorly understood. Here, we report that docosahexaenoic acid (DHA) robustly induced autophagy in murine bone marrow-derived macrophages (BMDMs). Treatment of T. gondii-infected macrophages with DHA resulted in colocalization of Toxoplasma parasitophorous vacuoles with autophagosomes and reduced intracellular survival of T. gondii. The autophagic and anti-Toxoplasma effects induced by DHA were mediated by AMP-activated protein kinase (AMPK) signaling. Importantly, BMDMs isolated from Fat-1 transgenic mice, a well-known animal model capable of synthesizing ω3-PUFAs from ω6-PUFAs, showed increased activation of autophagy and AMPK, leading to reduced intracellular survival of T. gondii when compared with wild-type BMDMs. Moreover, Fat-1 transgenic mice exhibited lower cyst burden in the brain following infection with the avirulent strain ME49 than wild-type mice. Collectively, our results revealed mechanisms by which endogenous ω3-PUFAs and DHA control T. gondii infection and suggest that ω3-PUFAs might serve as therapeutic candidate to prevent toxoplasmosis and infection with other intracellular protozoan parasites.

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we developed a zebrafish infection model to study the innate immune response to Toxoplasma in vivo. By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision-driven automated image analysis, we reveal that Toxoplasma invades brain cells and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We also show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are cleared in vivo, we further analyzed Toxoplasma-macrophage interactions using time-lapse microscopy and three-dimensional correlative light and electron microscopy (3D CLEM). Time-lapse microscopy revealed that macrophages are recruited to the infection site and play a key role in Toxoplasma control. High-resolution 3D CLEM revealed parasitophorous vacuole breakage in brain cells and macrophages in vivo, suggesting that cell-intrinsic mechanisms may be used to destroy the intracellular niche of tachyzoites. Together, our results demonstrate in vivo control of Toxoplasma by macrophages, and highlight the possibility that zebrafish may be further exploited as a novel model system for discoveries within the field of parasite immunity. This article has an associated First Person interview with the first author of the paper.

Toxoplasma gondii is a protozoan parasite of medical and veterinary significance that is able to infect any warm-blooded vertebrate host. In addition to its importance to public health, several inherent features of the biology of T. gondii have made it an important model organism to study host–pathogen interactions. One factor is the genetic tractability of the parasite, which allows studies on the microbial factors that affect virulence and allows the development of tools that facilitate immune studies. Additionally, mice are natural hosts for T. gondii , and the availability of numerous reagents to study the murine immune system makes this an ideal experimental system to understand the functions of cytokines and effector mechanisms involved in immunity to intracellular microorganisms. In this article, we will review current knowledge of the innate and adaptive immune responses required for resistance to toxoplasmosis, the events that lead to the development of immunopathology, and the natural regulatory mechanisms that limit excessive inflammation during this infection.

Toxoplasma gondii (T. gondii), a prolific protozoan parasite, forms cysts within neurons of the central nervous system that maintain infection for the lifetime of the host. Astrocytes are fundamental to neuronal health by providing nutrients and structural support and help regulate neurotransmitters by continuous communication with neurons. It is not yet known how infection and the presence of intracellular cysts, disrupts the crucial relationship between these cells. Extracellular vesicles (EVs) function in intracellular communication and can contain proteins, lipids, DNA, miRNA, and other RNA subtypes. EVs are produced by all cells and play an important role in neuronal-astrocyte interactions, including the regulation of glutamate receptors on astrocytes. Previous work has demonstrated that Toxoplasma infection reduces astrocytic expression of the primary glutamate transporter, GLT-1. Here we tested if cyst infection of neurons alters the production and content of EVs. EVs were isolated from uninfected and infected primary murine cortical neurons and their size, concentration, and characterization were confirmed with nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), ELISA, western blot, liquid chromatography (LC)-mass spectrometry (MS)/MS, and microRNA sequencing. Analysis reveals that infection of neurons reduced neuronal production of EVs and altered their protein and miRNA content. In addition to changes in host protein content, EVs from infected neurons contained the Toxoplasma proteins GRA1, GRA2, GRA7, MAG1 and MAG2. Following incubation of neuronal EVs with primary astrocytes, GRA7 protein could be observed within intracellular EVs and the nuclei of GRA7  +  EV-containing cells. EVs from infected neurons altered gene expression of astrocytes resulting in an increase in pro-inflammatory transcriptional signatures, along with a downregulation of GLT-1 protein expression with similar transcriptional changes found in astrocytes in vivo . These results demonstrate the ability of a parasitic infection in the brain to alter EV production and the fundamental communication between neurons and astrocytes.

Genome-wide CRISPR screening is a powerful tool to identify genes required under selective conditions. However, the inherent scale of genome-wide libraries can limit their application in experimental settings where cell numbers are restricted, such as in vivo infections or single cell analysis. The use of small scale CRISPR libraries targeting gene subsets circumvents this problem. Here we develop a method for rapid generation of custom guide RNA (gRNA) libraries using arrayed single-stranded oligonucleotides for reproducible pooled cloning of CRISPR/Cas9 libraries. We use this system to generate mutant pools of different sizes in the protozoan parasite Toxoplasma gondi and describe optimised analysis methods for small scale libraries. An in vivo genetic screen in the murine host identifies novel and known virulence factors and we confirm results using cloned knock-out parasites. Our study also reveals a potential trans-rescue of individual knock-out parasites in pools of mutants compared to homogenous knock-out lines of the key virulence factor MYR1. Targeted CRISPR libraries expand the use of genetic screens across experimental conditions. Here, the authors develop a method for generating and analysing small scale custom CRISPR libraries and use it in the human and livestock pathogen Toxoplasma gondii to identify virulence factors in mice.

Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival. The destruction of PVs by IFNγ-inducible guanylate binding protein (GBP) and immunity-related GTPase (IRG) host proteins is central to a successful immune response directed against numerous PV-resident pathogens. However, the mechanism bywhich IRGs and GBPs cooperatively detect and destroy PVs is unclear. We find that host cell priming with IFNγ prompts IRG-dependent association ofToxoplasma-andChlamydia-containing vacuoles with ubiquitin through regulated translocation of the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). This initial ubiquitin labeling elicits p62-mediated escort and deposition of GBPs to PVs, thereby conferring cell-autonomous immunity. Hypervirulent strains ofToxoplasma gondiievade this process via specific rhoptry protein kinases that inhibit IRG function, resulting in blockage of downstream PV ubiquitination and GBP delivery. Our results define a ubiquitin-centered mechanism by which host cells deliver GBPs to PVs and explain how hypervirulent parasites evade GBP-mediated immunity.