Toxoplasmosis is a significant global zoonosis with devastating impacts, and an effective vaccine against toxoplasmosis for humans has not yet been developed. In this study, we designed and formulated a novel DNA vaccine encoding the inhibitor of STAT1 transcriptional activity (IST) of T. gondii utilizing the eukaryotic expression vector pEGFP-N1 for the first time, with CL264 being a molecular adjuvant. Following intramuscular injection of the vaccine into mice, the levels of antibodies and cytokines were assessed to evaluate the immune response. Additionally, mice were challenged with highly virulent RH-strain tachyzoites of T. gondii, and their survival time was observed. The results show that the levels of IgG in serum, the ratio of IgG2a/IgG1 and the levels of IFN-γ in splenocytes of mice were significantly higher in the pEGFP-TgIST group and the pEGFP-TgIST + CL264 group than in the control group. In addition, the proportion of CD4+/CD8+ T cells was higher in mice immunized with either the pEGFP-TgIST group (p < 0.001) or the pEGFP-TgIST + CL264 group (p < 0.05) compared to the three control groups. Notably, TgIST-immunized mice exhibited prolonged survival times after T. gondii RH strain infection (p < 0.05). Our findings collectively demonstrate that the TgIST DNA vaccine elicits a significant humoral and cellular immune response and offers partial protection against acute T. gondii infection in the immunized mice, which suggests that TgIST holds potential as a candidate for further development as a DNA vaccine.

TORCH infections usually result in mild maternal morbidity, but may cause severe congenital abnormalities. Therefore, it is important to detect maternal infections, monitor the fetus after the disease has been recognized, and define the seronegative women who are at risk of primary infection during pregnancy. From 2014 to 2023, serum samples from 1032 childbearing-aged and pregnant women (16-45 years) were tested for IgM/IgG antibodies to the most common TORCH pathogens: Toxoplasma gondii, rubella virus (RUBV), cytomegalovirus (CMV), and herpes simplex viruses (HSV-1 and HSV-2). The overall IgG seroprevalence rates were 20.1% for T. gondii, 91.3% for RUBV, 70.5% for CMV, 66.8% for HSV-1, and 3.5% for HSV-2. Only HSV-2 seroprevalence was age-related, with a significant progressive increase in seropositivity from 0% in those aged less than 26 years to 9.3% in those older than 40 years. The seroprevalence of T. gondii was higher in residents of suburban/rural areas than in residents of urban areas (27.4% vs. 17.1%). In addition, participants from continental regions were more often toxoplasma-seropositive than those from coastal regions (22.2% vs. 15.3%). HSV-1 seroprevalence was also higher in suburban/rural areas (71.7% vs. 64.7%). Obstetric history was not associated with TORCH seropositivity. Univariate and multivariate risk analysis showed that suburban/rural areas of residence and continental geographic regions were significant risk factors for T. gondii seroprevalence. Furthermore, suburban/rural area of residence was a significant risk factor for HSV-1 seroprevalence, while older age was a significant risk factor for HSV-2 seroprevalence. A declining trend in the seroprevalence of all TORCH pathogens was observed compared to previous Croatian studies (2005-2011). Similarly, the proportion of women simultaneously IgG-seropositive to two or three pathogens decreased over time. The maternal serology before pregnancy could potentially reduce the burden of congenital TORCH infections.

UNASSIGNED: Soil is an appropriate substrate for the storage and transmission of oocytes of Toxoplasma gondii. Ingestion of soil contaminated with T. gondii oocysts is a major transmission route of human and animal toxoplasmosis. The present study was carried out to investigate soil contamination with T. gondii oocysts in urban and rural areas of Guilan Province, northern Iran.
UNASSIGNED: Overall, 208 soil samples were collected from 16 cities and villages in Guilan Province, northern Iran from Oct 2020 to Nov 2021. Soil samples were investigated using modified sucrose flotation technique. Real-time polymerase chain reaction was used to detect presence of T. gondii DNAs in the samples. Positive samples were further analyzed using nested polymerase chain reaction for GRA6 gene. Moreover, six selected positive samples were used for amplifying and sequencing of the GRA6 gene.
UNASSIGNED: Overall, 31 samples were positive for T. gondii with frequency of 14.9% and ranging from 10.9% in rural areas to 16.3% in urban areas. Statistical analysis showed significant differences between the seasons (P=0.003). The phylogenetic analysis illustrated that our six sequences were similar and closely related to Type I strain of T. gondii.
UNASSIGNED: Results showed relatively high levels (14.9%) of T. gondii oocytes in soil samples of Guilan Province, northern Iran, which provided essential data for the effective prevention and control of toxoplasmosis in the region.

Toxoplasma gondii and Neospora caninum are major worldwide morbidity-causing pathogens. Bumped kinase inhibitors (BKIs) are a compound class that has been optimized to target the apicomplexan calcium-dependent protein kinase 1 (CDPK1) - and several members of this class have proven to be safe and highly active in vitro and in vivo. BKI-1708 is based on a 5-aminopyrazole-4-carboxamide scaffold, and exhibited in vitro IC50 values of 120 nM for T. gondii and 480 nM for N. caninum β-galactosidase expressing strains, and did not affect human foreskin fibroblast (HFF) viability at concentrations up to 25 μM. Electron microscopy established that exposure of tachyzoite-infected fibroblasts to 2.5 μM BKI-1708 in vitro induced the formation of multinucleated schizont-like complexes (MNCs), characterized by continued nuclear division and harboring newly formed intracellular zoites that lack the outer plasma membrane. These zoites were unable to finalize cytokinesis to form infective tachyzoites. BKI-1708 did not affect zebrafish (Danio rerio) embryo development during the first 96 h following egg hatching at concentrations up to 2 μM. Treatments of mice with BKI-1708 at 20 mg/kg/day during five consecutive days resulted in drug plasma levels ranging from 0.14 to 4.95 μM. In vivo efficacy of BKI-1708 was evaluated by oral application of 20 mg/kg/day from day 9-13 of pregnancy in mice experimentally infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. This resulted in significantly decreased cerebral parasite loads and reduced vertical transmission in both models without drug-induced pregnancy interference.

UNASSIGNED: Wildlife represents an increasingly important source of pathogens of medical and veterinary importance. Surveillance in wildlife offers an insight on current epidemiological status of selected pathogens and help to prevent spillovers to humans and livestock.
UNASSIGNED: Our study included 312 wild ruminants belonging to five species: Roe deer (n = 134), red deer (n = 113), Alpine chamois (n = 53), European mouflon (n = 10) and Alpine ibex (n = 2). Seven pathogens that may have profound effect on human/livestock health and economic viability of the farms were tested using serological methods.
UNASSIGNED: Antibodies against Toxoplasma gondii, Neospora caninum, Coxiella burnetii, Brucella spp., Chlamydophila abortus, Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium bovis were detected in 34.62% (108/312), 0.96% (3/312), 2.24% (7/312), 0, 0.96% (3/312), 0, 0.64% (2/312) of animals tested, respectively. Because of low prevalences, risk factors were assessed only for T. gondii. Sex (female>male) and species (roe deer>red deer, roe deer>Alpine chamois) were significantly associated with the T. gondii positive outcome, while age was not.
UNASSIGNED: Adult males had the lowest T. gondii prevalence which offers future research opportunities. The lower seroprevalence of most investigated pathogens suggests game meat, if properly cooked, as being relatively safe for human consumption. This is the first study investigating the seroprevalence and associated risk factors of selected pathogens in wild ruminants in Slovenia.

UNASSIGNED: Toxoplasma gondii is an intracellular parasite of importance to human and veterinary health. The structure and diversity of the genotype population of T. gondii varies considerably with respect to geography, but three lineages, type I, II and III, are distributed globally. Lineage III genotypes are the least well characterized in terms of biology, host immunity and virulence. Once a host is infected with T.gondii, innate immune mechanisms are engaged to reduce the parasite burden in tissues and create a pro-inflammatory environment in which the TH1 response develops to ensure survival. This study investigated the early cellular immune response of Swiss-Webster mice post intraperitoneal infection with 10 tachyzoites of four distinct non-clonal genotypes of lineage III and a local isolate of ToxoDB#1. The virulence phenotype, cumulative mortality (CM) and allele profiles of ROP5, ROP16, ROP18 and GRA15 were published previously.
UNASSIGNED: Parasite dissemination in different tissues was analyzed by real-time PCR and relative expression levels of IFNγ, IL12-p40, IL-10 and TBX21 in the cervical lymph nodes (CLN), brain and spleen were calculated using the ΔΔCt method. Stage conversion was determined by detection of the BAG1 transcript in the brain.
UNASSIGNED: Tissue dissemination depends on the virulence phenotype but not CM, while the TBX21 and cytokine levels and kinetics correlate better with CM than virulence phenotype. The earliest detection of BAG1 was seven days post infection. Only infection with the genotype of high CM (69.4%) was associated with high T-bet levels in the CLN 24 h and high systemic IFNγ expression which was sustained over the first week, while infection with genotypes of lower CM (38.8%, 10.7% and 6.8%) is characterized by down-regulation and/or low systemic levels of IFNγ. The response intensity, as assessed by cytokine levels, to the genotype of high CM wanes over time, while it increases gradually to genotypes of lower CM.
UNASSIGNED: The results point to the conclusion that the immune response is not correlated with the virulence phenotype and/or allele profile, but an early onset, intense pro-inflammatory response is characteristic of genotypes with high CM. Additionally, high IFNγ level in the brain may hamper stage conversion.

UNASSIGNED: Toxoplasma gondii, a neurotropic protozoan, infects up one to third of the world population. The parasite can invade a wide variety of nucleated cells but preferably glial cells. Glia maturation factor β (GMFβ), a 17KD protein expressed at high levels in the central nervous system is predominantly related to neurodegenerative diseases such as Alzheimer\'s disease, Parkinson\'s disease, and Multiple sclerosis. We aimed to determine the expression level of GMFβ and its relation to other pro-inflammatory factors (IL33, SDF1, and CCL2) on T. gondii infected human neuroblastoma cell line.
UNASSIGNED: The human neuroblastoma (SK_NMC C535) cell line was infected by 5×106 (1:1 ratio). The supernatant was collected after cell lysis and centrifugation. Total RNA was extracted using the Yekta Tajhiz RNA extraction kit. cDNA was synthesized based on RevertAid First Strand cDNA Synthesis Kit manufacturer\'s protocol (Parstous, cDNA synthesis kit, Iran). The specificity of each primer pair (GMFβ, IL33, SDF1, and CCL2) was provided by NCBI BLAST. Gene expression level was measured using Real-Time PCR. All experiments were conducted at the Hamadan University of Medical Sciences, western Iran in 2022.
UNASSIGNED: The GMFβ increased significantly up to 1.35-fold (P=0.007). The increase in GMFβ expression in neuroblastoma cells was consistent with the increase in pro-inflammatory factors (CCL2 (0.47), IL33 (0.152) and, SDF1 (1.33)).
UNASSIGNED: GMFβ upregulation can be a novel indicator of the destruction of nerve cells.

The placenta plays a critical role in host-pathogen interactions. Thus, ex vivo infection of mammalian placental explants is an excellent and simple method to study the mechanisms of cellular and tissue invasion by different pathogens in different mammalian species. These explants can be maintained in culture for several days, preserving the tissue architecture and resembling in-utero conditions under more physiological conditions than their isolated counterparts in isolated cell culture models. In addition, placental explants not only allow us to study how the placenta responds and defends itself against various infections but also provide a versatile platform for advancing our understanding of placental biology and the immune response. Furthermore, they serve as powerful tools for drug discovery, facilitating the screening of potential therapeutics for placental infections and for the identification of diagnostic markers. This review highlights the utility of mammalian placental explants in studying the host-pathogen interaction of two relevant protozoan parasites, Trypanosoma cruzi, the causative agent of Chagas disease, and Toxoplasma gondii, the etiological agent of Toxoplasmosis. Here, we discuss the different methodologies and technical aspects of the model, as well as the effect of both parasites on placental responses in human, canine, and ovine explants.

Toxoplasma gondii is an opportunistic pathogen that can intrude into the blood-brain barrier and reside in the brain only with low inflammatory reaction. When infected with HIV, the immune system becomes severely compromised and leads to the reactivation of latent toxoplasmosis infection, which can mimic the clinical manifestation of stroke. We report a case of a 65-year-old female patient who presented with sudden right limb weakness, walking difficulty, and numbness without other typical symptoms, raising suspicion of acute ischemic stroke. The HIV serology returned positive, which expedited the diagnostic workup for opportunistic infection. Combining imageological examination and metagenomics next-generation sequencing of cerebrospinal fluid, HIV-associated cerebral toxoplasmosis was confirmed. The patient underwent treatment for toxoplasmosis and HIV. Six months after onset, the patient can walk independently but still exhibits weakness in the right upper limb. In HIV-infected patients, cerebral toxoplasmosis, particularly presenting as isolated stroke-like episodes, poses a more significant challenge, emphasizing the need for more thorough investigations to reduce the potential for misdiagnosis.

Toxoplasmosis is an important zoonotic disease caused by Toxoplasma gondii that can infect almost all warm-blooded animals worldwide, including humans. The high prevalence of T. gondii infection and its ability to cause serious harm to humans and animals, especially immunodeficient individuals, make it a key public health issue. Accurate diagnostic tools with high sensitivity are needed for controlling T. gondii infection. In the current study, we compared the performance of recombinant SAG2, GRA6, and GRA7 in ELISA for the serological diagnosis of T. gondii infection in cats. We further investigated the antigenicity of recombinant dense granule protein 3 (rGRA3), rGRA5, rGRA8, and rSRS29A expressed in a plant-based, cell-free expression system for detecting antibodies in T. gondii-infected cats. In summary, our data suggest that GRA7 is more sensitive than the other two antigens for the serodiagnosis of T. gondii infection in cats, and GRA3 expressed in the cell-free system is also a priming antigen in serological tests for detecting T. gondii infection in cats.