Fam96a is essential for the host control of Toxoplasma gondii infection by fine-tuning macrophage polarization via an iron-dependent mechanism.

BACKGROUND: Toxoplasmosis affects a quarter of the world's population. Toxoplasma gondii (T.gondii) is an intracellular parasitic protozoa. Macrophages are necessary for proliferation and spread of T.gondii by regulating immunity and metabolism. Family with sequence similarity 96A (Fam96a; formally named Ciao2a) is an evolutionarily conserved protein that is highly expressed in macrophages, but whether it play a role in control of T. gondii infection is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we utilized myeloid cell-specific knockout mice to test its role in anti-T. gondii immunity. The results showed that myeloid cell-specific deletion of Fam96a led to exacerbate both acute and chronic toxoplasmosis after exposure to T. gondii. This was related to a defectively reprogrammed polarization in Fam96a-deficient macrophages inhibited the induction of immune effector molecules, including iNOS, by suppressing interferon/STAT1 signaling. Fam96a regulated macrophage polarization process was in part dependent on its ability to fine-tuning intracellular iron (Fe) homeostasis in response to inflammatory stimuli. In addition, Fam96a regulated the mitochondrial oxidative phosphorylation or related events that involved in control of T. gondii. CONCLUSIONS/SIGNIFICANCE: All these findings suggest that Fam96a ablation in macrophages disrupts iron homeostasis and inhibits immune effector molecules, which may aggravate both acute and chronic toxoplasmosis. It highlights that Fam96a may autonomously act as a critical gatekeeper of T. gondii control in macrophages.

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.

Single-cell transcriptomic analysis of decidual immune cell landscape in the occurrence of adverse pregnancy outcomes induced by Toxoplasma gondii infection.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that can lead to adverse pregnancy outcomes, particularly in early pregnancy. Previous studies have illustrated the landscape of decidual immune cells. However, the landscape of decidual immune cells in the maternal-fetal microenvironment during T. gondii infection remains unknown. METHODS: In this study, we employed single-cell RNA sequencing to analyze the changes in human decidual immune cells following T. gondii infection. The results of scRNA-seq were further validated with flow cytometry, reverse transcription-polymerase chain reaction, western blot, and immunofluorescence staining. RESULTS: Our results showed that the proportion of 17 decidual immune cell clusters and the expression levels of 21 genes were changed after T. gondii infection. Differential gene analysis demonstrated that T. gondii infection induced the differential expression of 279, 312, and 380 genes in decidual NK cells (dNK), decidual macrophages (dMφ), and decidual T cells (dT), respectively. Our results revealed for the first time that several previously unknown molecules in decidual immune cells changed following infection. This result revealed that the function of maternal-fetal immune tolerance declined, whereas the killing ability of decidual immune cells enhanced, eventually contributing to the occurrence of adverse pregnancy outcomes. CONCLUSIONS: This study provides valuable resource for uncovering several novel molecules that play an important role in the occurrence of abnormal pregnancy outcomes induced by T. gondii infection.

Human T-cell activation with Toxoplasma gondii antigens loaded in maltodextrin nanoparticles.

Toxoplasmosis is the most prevalent parasitic zoonosis worldwide, causing ocular and neurological diseases. No vaccine has been approved for human use. We evaluated the response of peripheral blood mononuclear cells (PBMCs) to a novel construct of Toxoplasma gondii total antigen in maltodextrin nanoparticles (NP/TE) in individuals with varying infectious statuses (uninfected, chronic asymptomatic, or ocular toxoplasmosis). We analyzed the concentration of IFN-γ after NP/TE ex vivo stimulation using ELISA and the immunophenotypes of CD4+ and CD8+ cell populations using flow cytometry. In addition, serotyping of individuals with toxoplasmosis was performed by ELISA using GRA6-derived polypeptides. Low doses of NP/TE stimulation (0.9 µg NP/0.3 µg TE) achieved IFN-γ-specific production in previously exposed human PBMCs without significant differences in the infecting serotype. Increased IFN-γ expression in CD4+ effector memory cell subsets was found in patients with ocular toxoplasmosis with NP/TE but not with TE alone. This is the first study to show how T-cell subsets respond to ex vivo stimulation with a vaccine candidate for human toxoplasmosis, providing crucial insights for future clinical trials.

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

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

Attenuated Toxoplasma gondii enhances the antitumor efficacy of anti-PD1 antibody by altering the tumor microenvironment in a pancreatic cancer mouse model.

PURPOSE: To investigate whether attenuated Toxoplasma is efficacious against solid tumors of pancreatic cancer and whether attenuated Toxoplasma improves the antitumor activity of αPD-1 antibody on pancreatic cancer. METHODS: The therapeutic effects of attenuated Toxoplasma NRTUA strain monotherapy and combination therapy of NRTUA with anti-PD-1 antibody on PDAC tumor volume and tumor weight of Pan02 tumor-bearing mice were investigated. We characterized the effects of combination therapy of NRTUA with anti-PD-1 antibody on tumor-infiltrating lymphocytes and tumor-specific IFN-γ by using immunohistochemistry, flow cytometry and ELISA. The antitumor mechanisms of combination therapy of NRTUA with anti-PD-1 antibody were investigated via depletion of CD8+ T cells and IL-12. RESULTS: NRTUA strain treatment inhibited tumor growth in a subcutaneous mouse model of PDAC through activating dendritic cells and increasing CD8+ T cell infiltration in the tumor microenvironment. More importantly, combination therapy of NRTUA with anti-PD-1 antibody elicited a significant antitumor immune response and synergistically controlled tumor growth in Pan02 tumor-bearing mice. Specifically, the combination treatment led to elevation of CD8+ T cell infiltration mediated by dendritic cell-secreted IL-12 and to tumor-specific IFN-γ production in the PDAC tumor microenvironment. Also, the combination treatment markedly reduced the immunosuppressive myeloid-derived suppressor cell population in PDAC mice. CONCLUSION: These findings could provide a novel immunotherapy approach to treating solid tumors of PDAC and overcoming resistance to anti-PD-1 agents in PDAC tumors.

Serological survey of Neospora caninum and Toxoplasma gondii in shelter-housed cats infected with feline immunodeficiency virus, Brazil / Inquérito sorológico para Neospora caninum e Toxoplasma gondii em um abrigo de gatos infectados com o vírus da imunodeficiência felina, Brasil

Felines play a leading role in the epidemiology of Toxoplasma gondii infection, but there is scarce information about the epidemiology of Neospora caninum, particularly in feline immunodeficiency virus (FIV)-infected cats. Cats seropositive to T. gondii do not usually show symptoms unless they are immunosuppressed, such as FIV-infected cats. The same relationship remains poorly known for N. caninum, although it has been associated with neurological disorders in HIV-infected people. Since FIV-infected cats are prone to develop encephalitis of unknown etiology, this study aimed to evaluate the presence of specific antibodies to T. gondii and N. caninum in a shelter for stray cats naturally infected with FIV. A total of 104 serum samples from cats living in a shelter, located in São Paulo city (Brazil), was assessed for T. gondii and N. caninum specific antibody by indirect fluorescent-antibody test (IFAT). Of the 104 cats, 25 (24%) were infected with FIV and, aside from these, 8 (32%) had antibodies against T. gondii (titers from 16 to 128). Only 1 (4%) of the FIV-infected cats had antibodies against N. caninum, which was the first record of coinfection. Among the FIV-naïve cats, 11 (14%) were positive for T. gondii(titers from 16 to 256) and only 1 (1.2%) had antibodies against N. caninum. Serologically positive reactions to T. gondii and N. caninum were not correlated with age or sex (p>0.05), and there was no correlation between FIV and the occurrence of anti-T. gondii or anti-N. caninum antibodies (p>0.05). Further studies encompassing larger cat populations from different origins and locations are essential to clarify the prevalence of T. gondii and N. caninum antibodies in FIV-positive cats.(AU)

Os felinos têm um papel importante na epidemiologia da infecção por Toxoplasma gondii, mas pouco se sabe sobre a epidemiologia da infecção por Neospora caninum em gatos, particularmente em gatos infectados com o vírus da imunodeficiência felina (FIV). Gatos soropositivos para Toxoplasma gondii geralmente não apresentam sintomas a não ser que estejam imunossuprimidos, como gatos infectados com FIV. A mesma relação ainda é pouco conhecida para N. caninum, embora tenha sido associada a distúrbios neurológicos em pessoas infectadas pelo HIV. Considerando que gatos infectados com FIV são propensos a desenvolver encefalite de etiologia desconhecida, o presente estudo teve como objetivo avaliar a presença de anticorpos específicos para T. gondii e N. caninum em gatos infectados com FIV. Um total de 104 amostras de soro de gatos residentes em um abrigo na cidade de São Paulo, Brasil, foram avaliadas para a presença de anticorpos contra T. gondii e N. caninum pelo teste de imunofluorescência indireta (RIFI). Dos 104 gatos, 25 (24%) estavam infectados com FIV e destes 8, (32%) tinham anticorpos contra T. gondii (titulação entre 16 e 128). Apenas 1 (4%) dos gatos infectados com FIV apresentava anticorpos contra N. caninum, sendo este o primeiro registro dessa coinfecção. Entre os gatos não infectados com FIV, 11 (14%) foram positivos para T. gondii (titulação entre 16 e 256) e apenas 1 (1,2%) tinha anticorpos contra N. caninum. A reação sorologicamente positiva para T. gondii e N. caninum não foi correlacionada com a idade ou sexo (p> 0,05), nem houve correlação entre FIV e ocorrência de anticorpos para T. gondii ou N. caninum(p> 0,05). Estudos subsequentes abrangendo populações maiores de gatos de diferentes origens e locais são essenciais para esclarecer a prevalência de anticorpos contra T. gondii e N. caninum em animais acometidos por FIV.(AU)

Toxoplasma gondii ROP18I inhibits host innate immunity through cGAS-STING signaling.

Toxoplasma gondii is an opportunistic protozoan, which widely infects humans and other warm-blooded animals. The type I interferon (IFN) such as IFN-α/ß is involved in cGAS-STING signaling to resist T. gondii infection. We found in RAW264.7 cells, that T. gondii virulence factor TgROP18I , inhibited IFN-ß production through interacting with interferon regulatory factor 3 (IRF3). Besides, TgROP18I interacted with p62 and Tumor Necrotic Factor Receptor Associated Factor 6 (TRAF6), which resulted in the inhibition of TRAF6-p62 interaction, and phosphorylation of p62. Furthermore, TgROP18I restricted the recruitment of ubiquitin, p62 and microtubule-associated protein light chain 3 (LC3) to the parasitophorous vacuole membrane (PVM) in IFN-γ-stimulated murine cell line L929 cells. In IFN-γ-stimulated human cells, TgROP18I restricted the decoration of PVM with ubiquitin, p62, and LC3, and bound with TRAF2, TRAF6, and p62, respectively. As a result, TgROP18I led to a successful parasitic replication in murine and human cells. Collectively, our study revealed the function of TgROP18I in suppressing host type I interferon responses in T. gondii infection for parasitic immune escape.

Seroprevalence and associated risk factors of Toxoplasma gondii and Neospora caninum infections in cattle in Central India.

Toxoplasma gondii and Neospora caninum are closely related cyst-forming parasites identified as important causes of reproductive failures in ruminants. While these parasites have been reported worldwide, seroprevalence and associated risk factors for cattle infections have not been determined in India. A total of 576 serum samples of cattle were analyzed for antibodies to T. gondii and N. caninum using enzyme-linked immunosorbent assay (ELISA), modified/Neospora agglutination test (MAT/NAT), and an indirect fluorescent antibody test (IFAT-tachyzoite and bradyzoite). Additionally, general information about cattle, movement of cats and dogs, the menace of rodents, management, and reproductive disorders were assessed to identify the potential risk factors. Overall, 32.9% (190/576) serum samples reacted positively to T. gondii and 24.8% (143/576) to N. caninum. The performance of the diagnostic tests showed excellent agreement between IFAT and ELISA (kappa [κ] = 0.98) and between MAT/NAT and ELISA (κ = 0.97). Combining both infections on avidity test, 94% sera had high-IgG avidity, and 3% had low-IgG avidity antibodies, indicating chronic infection in the majority of the cases. The identified risk factors (p < 0.05) for exposure to T. gondii were: increasing age (Odds Ratio [OR]: 2.02), movement of cat (OR: 4.8) and rodents (OR: 1.57) in the farm; and for N. caninum: increasing age (OR: 1.6), movement of dogs in the farm (OR: 2.07), drinking pond water (OR: 1.64) and abortion (OR: 1.82). These findings revealed that T. gondii and N. caninum infections are widespread in the study area and suggest conducting nationwide epidemiological studies owing to their economic importance.

Macrophage Stimulation as a Useful Approach for Immunoscreening of Potential Vaccine Candidates Against Toxoplasma gondii and Neospora caninum Infections.

Toxoplasmosis and neosporosis are protozoan diseases that adversely affect the medical and additionally veterinary sectors, respectively. Toxoplasmosis is caused by Toxoplasma gondii which infects almost all warm-blooded animals including humans. While, neosporosis is caused by Neospora caninum, which induces infection in many animal species particularly in cattle. Currently, control measures for both infections are defective because of no effective vaccine or treatment. Macrophages constitute the first line of innate immunity, which contributes to the effective elimination of T. gondii or N. caninum. This action is mediated by IL-12, which is critical for the secretion of interferon gamma (IFN-γ). Successful vaccine candidates against both protozoan parasites should possess the ability to induce the cellular immune response and IFN-γ production. In this chapter, we will focus on an efficient immunological approach for discovery of potential vaccine candidates against above-mentioned parasites. Our previous studies revealed a strong correlation between vaccine antigens that enhanced the macrophage secretion of IL-12 and their efficacy as potential vaccine candidates in murine model. In case of T. gondii, peroxiredoxin 1 (TgPrx1) and peroxiredoxin 3 stimulated the production of IL-12 from murine peritoneal macrophages and conferred strong to moderate protection in C57BL/6 mice, respectively. At the same context, Neospora antigens of dense granule protein 6 (NcGRA6) and cyclophilin entrapped with oligo-mannose coated-liposomes stimulated macrophage IL-12 secretion and substantially protected immunized BALB/c mice. Therefore, we can deduce that macrophage stimulation evidenced in IL-12 production can be used as a useful approach for judgment of vaccine efficacy before further evaluation using in vivo experiments. Methods of vaccine preparation and macrophage stimulation will be fully described for TgPrx1 and NcGRA6 as potential vaccine candidates against toxoplasmosis and neosporosis, respectively.

Toxoplasma-proximal and distal control by GBPs in human macrophages.

Human guanylate binding proteins (GBPs) are key players of interferon-gamma (IFNγ)-induced cell intrinsic defense mechanisms targeting intracellular pathogens. In this study, we combine the well-established Toxoplasmagondii infection model with three in vitro macrophage culture systems to delineate the contribution of individual GBP family members to control this apicomplexan parasite. Use of high-throughput imaging assays and genome engineering allowed us to define a role for GBP1, 2 and 5 in parasite infection control. While GBP1 performs a pathogen-proximal, parasiticidal and growth-restricting function through accumulation at the parasitophorous vacuole of intracellular Toxoplasma, GBP2 and GBP5 perform a pathogen-distal, growth-restricting role. We further find that mutants of the GTPase or isoprenylation site of GBP1/2/5 affect their normal function in Toxoplasma control by leading to mis-localization of the proteins.

Detection of Sarcocystis spp. and Toxoplasma gondii in swine and detection of DNA of these protozoa in tissues and sausages / Soroprevalência de Sarcocystis spp. e Toxoplasma gondii em suínos e detecção do DNA desses protozoários em tecidos e embutidos

Abstract The seroprevalence of Sarcocystis spp. and Toxoplasma gondii was researched in swine raised in Santa Maria, RS, Brazil. Serum samples from 84 pigs from 31 farms were tested using indirect immunofluorescence assay (IFA) for both agents. Additionally, 53 samples of pork sausages and tissues destined for human consumption, including: salami, sausage, black pudding, heart, tongue, brain, and rib muscle, were submitted to PCR to detect DNA for each agent. The frequency of anti-Sarcocystis spp. antibodies was 36.9% (31/84), with titers ranging from 32 to 1024, and 25% (21/84) for anti-T. gondii antibodies, with titers ranging from 64 to 2048. Sarcocystis spp. and T. gondii DNA were detected in 67.9% (36/53) and 13.2% (7/53) of samples, respectively. The presence of antibodies and the detection of DNA from Sarcocystis spp., and T. gondii suggests that the pigs were infected and may serve as an important reservoir for both parasites. The infection by these protozoa in the swine population is relevant to public health due to their zoonotic potential.

Resumo A soroprevalência de Sarcocystis spp. e Toxoplasma gondii foi pesquisada em suínos criados em Santa Maria, RS, Brasil. Amostras de soro de 84 suínos de 31 fazendas foram testadas pela reação deimunofluorescência indireta (IFA) para ambos os agentes. Adicionalmente, 53 amostras de embutidos suínos e tecidos cárneos destinados ao consumo humano, incluindo: salame, linguiça, morcela, coração, língua, cérebro e músculo da costela foram submetidas à PCR para detecção de DNA para cada agente. A frequência de anticorpos anti-Sarcocystis spp. foi de 36,9% (31/84), com títulos variando de 32 a 1.024; e 25% (21/84) para anticorpos anti-T. gondii, com títulos variando de 64 a 2048. A presença de DNA de Sarcocystis spp. e T. gondii foi detectada em 67,9% (36/53) e 13,2% (7/53) das amostras avaliadas, respectivamente. A detecção de anticorpos e DNA de Sarcocystis spp. e T. gondii sugere que os suínos foram infectados e podem servir como um importante reservatório de ambos os parasitas. A circulação desses agentes na população suína é relevante para a saúde pública devido ao seu potencial zoonótico.

A uracil auxotroph Toxoplasma gondii exerting immunomodulation to inhibit breast cancer growth and metastasis.

BACKGROUND: Breast cancer is the most common cause of cancer-related death among women, and prognosis is especially poor for patients with triple-negative breast cancer (TNBC); therefore, there is an urgent need for new effective therapies. Recent studies have demonstrated that the uracil auxotroph Toxoplasma gondii vaccine displays anti-tumor effects. Here, we examined the immunotherapy effects of an attenuated uracil auxotroph strain of T. gondii against 4T1 murine breast cancer. METHODS: We constructed a uracil auxotroph T. gondii RH strain via orotidine 5'-monophosphate decarboxylase gene deletion (RH-Δompdc) with CRISPR/Cas9 technology. The strain's virulence in the T. gondii-infected mice was determined in vitro and in vivo by parasite replication assay, plaque assay, parasite burden detection in mice peritoneal fluids and survival analysis. The immunomodulation ability of the strain was evaluated by cytokine detection. Its anti-tumor effect was evaluated after its in situ inoculation into 4T1 tumors in a mouse model; the tumor volume was measured, and the 4T1 lung metastasis was detected by hematoxylin and eosin and Ki67 antibody staining, and the cytokine levels were measured by an enzyme-linked immunosorbent assay. RESULTS: The RH-Δompdc strain proliferated normally when supplemented with uracil, but it was unable to propagate without the addition of uracil and in vivo, which suggested that it was avirulent to the hosts. This mutant showed vaccine characteristics that could induce intense immune responses both in vitro and in vivo by significantly boosting the expression of inflammatory cytokines. Inoculation of RH-Δompdc in situ into the 4T1 tumor inhibited tumor growth, reduced lung metastasis, promoted the survival of the tumor-bearing mice and increased the secretion of Th1 cytokines, including interleukin-12 (IL-12) and interferon-γ (INF-δ), in both the serum and tumor microenvironment (TME). CONCLUSION: Inoculation of the uracil auxotroph RH-Δompdc directly into the 4T1 tumor stimulated anti-infection and anti-tumor immunity in mice, and resulted in inhibition of tumor growth and metastasis, promotion of the survival of the tumor-bearing mice and increased secretion of IL-12 and IFN-γ in both the serum and TME. Our findings suggest that the immunomodulation caused by RH-Δompdc could be a potential anti-tumor strategy.

Toxoplasma gondii-Induced Neutrophil Extracellular Traps Amplify the Innate and Adaptive Response.

Toxoplasmosis affects one-third of the human population worldwide. Humans are accidental hosts and are infected after consumption of undercooked meat and water contaminated with Toxoplasma gondii cysts and oocysts, respectively. Neutrophils have been shown to participate in the control of T. gondii infection in mice through a variety of effector mechanisms, such as reactive oxygen species (ROS) and neutrophil extracellular trap (NET) formation. However, few studies have demonstrated the role of neutrophils in individuals naturally infected with T. gondii. In the current study, we evaluated the activation status of neutrophils in individuals with acute or chronic toxoplasmosis and determined the role of T. gondii-induced NET formation in the amplification of the innate and adaptive immune responses. We observed that neutrophils are highly activated during acute infection through increased expression of CD66b. Moreover, neutrophils from healthy donors (HDs) cocultured with tachyzoites produced ROS and formed NETs, with the latter being dependent on glycolysis, succinate dehydrogenase, gasdermin D, and neutrophil elastase. Furthermore, we observed elevated levels of the chemokines (CXC motif) CXCL8 and (CC motif) CCL4 ligands in plasma from patients with acute toxoplasmosis and production by neutrophils from HDs exposed to T. gondii. Finally, we showed that T. gondii-induced NETs activate neutrophils and promote the recruitment of autologous CD4+ T cells and the production of interferon gamma (IFN-γ), tumor necrosis factor (TNF), interleukin 6 (IL-6), IL-17, and IL-10 by peripheral blood mononuclear cells. In conclusion, we demonstrated that T. gondii activates neutrophils and promotes the release of NETs, which amplify human innate and adaptive immune responses. IMPORTANCE Approximately one-third of the human population is estimated to be chronically infected with the obligate intracellular parasite Toxoplasma gondii. Humans are accidental hosts that are infected with T. gondii after consumption of undercooked meat or contaminated water. Neutrophils have been shown to control T. gondii growth by different mechanisms, including neutrophil extracellular traps (NETs). In the current study, we observed that neutrophils are highly activated during acute toxoplasmosis. We also determined that T. gondii-induced NETs are dependent on the energetic profile of neutrophils as well as the production of ROS and gasdermin D (GSDMD) cleavage. In addition, we showed that T. gondii-induced NETs activate neutrophils, promote the recruitment of autologous CD4+ T cells, and induce the production of cytokines by peripheral blood mononuclear cells, amplifying the innate and adaptive immune responses.

Characterization and evaluation of a recombinant multiepitope peptide antigen MAG in the serological diagnosis of Toxoplasma gondii infection in pigs.

BACKGROUND: Toxoplasmosis caused by Toxoplasma gondii is a serious disease threatening human and animal health. People can be infected with T. gondii by ingesting raw pork contaminated with cysts or oocysts. Serological test is a sensitive and specific method usually used for large-scale diagnosis of T. gondii infection in humans and animals (such as pigs). Commercial pig Toxoplasma antibody ELISA diagnostic kits are expensive, which limits their use; moreover, the wide antigen composition used in these diagnostic kits is still unclear and difficult to standardize. The multiepitope peptide antigen is a novel diagnostic marker, and it has potential to be developed into more accurate and inexpensive diagnostic kits. METHODS: The synthetic multiepitope antigen (MAG) cDNA encoding a protein with epitopes from five T. gondii-dominant antigens (SAG1, GRA1, ROP2, GRA4, and MIC3) was designed, synthesized, and expressed in Escherichia coli BL21 (DE3) strain. The recombinant protein was detected through western blot with pig anti-T. gondii-positive and -negative serum, and then IgG enzyme-linked immunosorbent assay (ELISA) named MAG-ELISA was designed. The MAG-ELISA was evaluated in terms of specificity, sensitivity, and stability. The MAG-ELISA was also compared with a commercial PrioCHECK® Toxoplasma Ab porcine ELISA (PrioCHECK ELISA). Finally, the trend of pig anti-T. gondii IgG levels after artificial infection with RH tachyzoites was evaluated using MAG-ELISA and two other ELISA methods (rMIC3-ELISA and PrioCHECK ELISA). RESULTS: MAG antigen could be specifically recognized by pig anti-T. gondii-positive but not -negative serum. MAG-ELISA showed high diagnostic performance in terms of specificity (88.6%) and sensitivity (79.1%). MAG-ELISA could be used for detecting anti-T. gondii IgG in the early stage of T. gondii infection in pigs (at least 7 days after artificial infection). CONCLUSIONS: Our results suggest that MAG antigen can be applied to specifically recognize anti-T. gondii IgG in pig, and MAG-ELISA has the potential for large-scale screening tests of T. gondii infection in pig farms and intensive industries.

P18 (SRS35/TgSAG4) Plays a Role in the Invasion and Virulence of Toxoplasma gondii.

Toxoplasmosis is a prevalent parasitic disease caused by Toxoplasma gondii (T. gondii). Under the control of the host immune system, T. gondii persists as latent bradyzoite cysts. Immunosuppression leads to their reactivation, a potentially life-threatening condition. Interferon-gamma (IFN-γ) controls the different stages of toxoplasmosis. Here, we addressed the role of the parasite surface antigen P18, belonging to the Surface-Antigen 1 (SAG-1) Related Sequence (SRS) family, in a cyst-forming strain. Deletion of P18 gene (KO P18) impaired the invasion of parasites in macrophages and IFN-γ-mediated activation of macrophages further reduced the invasion capacity of this KO, as compared to WT strain. Mice infected by KO P18, showed a marked decrease in virulence during acute toxoplasmosis. This was consequent to less parasitemia, accompanied by a substantial recruitment of dendritic cells, macrophages and natural killer cells (NK). Furthermore, KO P18 resulted in a higher number of bradyzoite cysts, and a stronger inflammatory response. A prolonged survival of mice was observed upon immunosuppression of KO P18 infected BALB/c mice or upon oral infection of Severe Combined Immunodeficiency (SCID) mice, with intact macrophages and natural killer (NK) cells. In stark contrast, oral infection of NSG (NOD/Shi-scid/IL-2Rγnull) mice, defective in macrophages and NK cells, with KO P18, was as lethal as that of the control strain showing that the conversion from bradyzoites to tachyzoites is intact and, suggesting a role of P18 in the response to host IFN-γ. Collectively, these data demonstrate a role for P18 surface antigen in the invasion of macrophages and in the virulence of the parasite, during acute and chronic toxoplasmosis.

Seroprevalence of Toxoplasma gondii and associated risk factors in domestic pigs raised from Cuba.

A cross-sectional study was carried out to determine the seroprevalence of Toxoplasma gondii and associated risk factors in pigs in the largest pork-producing region in Cuba. Serum samples from 420 pigs, including 210 sows and 210 post-weaning pigs, were tested for antibodies against T. gondii using a commercial indirect enzyme-linked immunosorbent assay. Anti-T. gondii antibodies were detected in 56 animals (13.3%, 95% CI: 10.1-16.6). A generalized estimating equations model revealed that the risk factors associated with higher seropositivity in pigs were altitude (higher in farm's location < 250 m above sea level (masl) versus ≥ 250 masl) and age (higher in sows compared to post-weaning pigs). The results indicated that this protozoan parasite is widely distributed on pig farms in the study area, which is a public health concern since the consumption of raw or undercooked pork meat products containing tissue cysts is considered one of the main routes of T. gondii transmission worldwide. Control measures should be implemented to reduce the risk of exposure to T. gondii in pigs in Cuba.

Burden and regional distribution of Toxoplasma gondii cysts in the brain of COBB 500 broiler chickens following chronic infection with 76K strain.

Toxoplasmosis is a worldwide zoonosis caused by the obligate intracellular apicomplexan parasite Toxoplasma gondii (T. gondii). Chickens are ground-feeders and represent, especially if free-range, important intermediate hosts in the epidemiology of toxoplasmosis and are used as sentinels of environmental contamination with T. gondii oocysts. Until now, little is known about the burden and regional distribution of T. gondii cysts in the chicken brain. It was therefore the aim of this study to investigate the abundance and specific distribution of T. gondii cysts within the chicken brain following chronic infection with a type II strain (76 K) of T. gondii. A total of 29 chickens were included in the study and divided into control group (n = 9) and two different infection groups, a low dose (n = 10) and a high dose (n = 10) group, which were orally inoculated with 1500 or 150,000 T. gondii oocysts per animal, respectively. Seroconversion was detected in the majority of chickens of the high dose group, but not in the animals of the low dose and the control group. Moreover, T. gondii DNA was detected most frequently in the brain and more frequently in the heart than in liver, spleen, thigh and pectoral muscle using qPCR analysis. The number of T. gondii cysts, quantified in the chicken brain using histological analysis, seems to be considerably lower as compared to studies in rodents, which might explain why T. gondii infected chickens very rarely, if at all, develop neurological deficits. Similar to observations in mice, in which no lateralisation for T. gondii cysts was reported, T. gondii cysts were distributed nearly equally between the left and right chicken brain hemispheres. When different brain regions (fore-, mid- and hindbrain) were compared, all T. gondii cysts were located in the forebrain with the overwhelming majority of these cysts being present in the telencephalic pallium and subpallium. More studies including different strains and higher doses of T. gondii are needed in order to precisely evaluate its cyst burden and regional distribution in the chicken brain. Together, our findings provide insights into the course of T. gondii infection in chickens and are important to understand the differences of chronic T. gondii infection in the chicken and mammalian brain.

Role of amylopectin synthesis in Toxoplasma gondii and its implication in vaccine development against toxoplasmosis.

Toxoplasma gondii is a ubiquitous pathogen infecting one-third of the global population. A significant fraction of toxoplasmosis cases is caused by reactivation of existing chronic infections. The encysted bradyzoites during chronic infection accumulate high levels of amylopectin that is barely present in fast-replicating tachyzoites. However, the physiological significance of amylopectin is not fully understood. Here, we identified a starch synthase (SS) that is required for amylopectin synthesis in T. gondii. Genetic ablation of SS abolished amylopectin production, reduced tachyzoite proliferation, and impaired the recrudescence of bradyzoites to tachyzoites. Disruption of the parasite Ca2+-dependent protein kinase 2 (CDPK2) was previously shown to cause massive amylopectin accumulation and bradyzoite death. Therefore, the Δcdpk2 mutant is thought to be a vaccine candidate. Notably, deleting SS in a Δcdpk2 mutant completely abolished starch accrual and restored cyst formation as well as virulence in mice. Together these results suggest that regulated amylopectin production is critical for the optimal growth, development and virulence of Toxoplasma. Not least, our data underscore a potential drawback of the Δcdpk2 mutant as a vaccine candidate as it may regain full virulence by mutating amylopectin synthesis genes like SS.

Seroprevalence and associated risk factors of Toxoplasma gondii infection among pregnant mother in Makassar, Indonesia.

The protozoan parasite, Toxoplasma gondii is estimated to infect one-third of the world's population. Infection in pregnant women can cause severe conditions for their babies. Until now, there is no data regarding Toxoplasma infection from Makassar pregnant mothers. This study aims to obtain information on Toxoplasma specific antibodies and to measure the risk factor associate with parasite infection. This cross-sectional study conducted in 9 of 47 primary health centres (Puskesmas) in Makassar. Blood samples and questionnaires were collected from 184 pregnant women aged 15-42 years old from September to October 2020. ELISA technique was used to examine the IgG and IgM antibodies. Univariable and multivariable analyses were carried out to measure factors that independently associate with Toxoplasma antibody positivity. Our result showed the range of Toxoplasma IgM and IgG are 0.06-1.01 and 0.09-3.01, respectively. While no one of our participants has an acute Toxoplasma gondii infection (IgM positive), we found 32,6% pregnant mothers are exposed to parasite (positive IgG). Contact with cats [OR(95%CI): 10.45(3.77-28.99)], consume chicken satay [OR(95%CI): 9.72(3.71-25.48)] and consume un-boiled water/ filtered water [OR(95%CI): 5.98(1.77-20.23)] are independently associate with positive Toxoplasma IgG antibody. Based on the result, we conclude that pregnant women in Makassar are exposed to T. gondii and the oocyst and tissue cyst of parasite contaminates food and water in Makassar.