Anti-Toxoplasma gondii effect of tylosin in vitro and in vivo.

BACKGROUND: Toxoplasma gondii is an important protozoan pathogen with medical and veterinary importance worldwide. Drugs currently used for treatment of toxoplasmosis are less effective and sometimes cause serious side effects. There is an urgent need for the development of more effective drugs with relatively low toxicity. METHODS: The effect of tylosin on the viability of host cells was measured using CCK8 assays. To assess the inhibition of tylosin on T. gondii proliferation, a real-time PCR targeting the B1 gene was developed for T. gondii detection and quantification. Total RNA was extracted from parasites treated with tylosin and then subjected to transcriptome analysis by RNA sequencing (RNA-seq). Finally, murine infection models of toxoplasmosis were used to evaluate the protective efficacy of tylosin against T. gondii virulent RH strain or avirulent ME49 strain. RESULTS: We found that tylosin displayed low host toxicity, and its 50% inhibitory concentration was 175.3 µM. Tylsoin also inhibited intracellular T. gondii tachyzoite proliferation, with a 50% effective concentration of 9.759 µM. Transcriptome analysis showed that tylosin remarkably perturbed the gene expression of T. gondii, and genes involved in "ribosome biogenesis (GO:0042254)" and "ribosome (GO:0005840)" were significantly dys-regulated. In a murine model, tylosin treatment alone (100 mg/kg, i.p.) or in combination with sulfadiazine sodium (200 mg/kg, i.g.) significantly prolonged the survival time and raised the survival rate of animals infected with T. gondii virulent RH or avirulent ME49 strain. Meanwhile, treatment with tylosin significantly decreased the parasite burdens in multiple organs and decreased the spleen index of mice with acute toxoplasmosis. CONCLUSIONS: Our findings suggest that tylosin exhibited potency against T. gondii both in vitro and in vivo, which offers promise for treatment of human toxoplasmosis.

Upregulation of lncRNA147410.3 in the Brain of Mice With Chronic Toxoplasma Infection Promoted Microglia Apoptosis by Regulating Hoxb3.

Toxoplasma gondii is neurotropic and affects the function of nerve cells, while the mechanism is unclear. LncRNAs are abundantly enriched in the brain and participated in the delicate regulation of the central nervous system (CNS) development. However, whether these lncRNAs are involved in the regulation of microglia activation during the process of T. gondii infection is largely unknown. In this study, the upregulation of a novel lncRNA147410.3 (ENSMUST00000147410.3) was identified as a key factor to influence this process. The target gene of lncRNA147410.3 was predicted and identified as Hoxb3. The localization of lncRNA147410.3 in the brain and cells was proved in the nucleus of neuroglia through FISH assay. Furthermore, the function of lncRNA147410.3 on neuronal cell was confirmed that lncRNA147410.3 could affect proliferation, differentiation, and apoptosis of mouse microglia by positively regulating Hoxb3. Thus, our study explored the modulatory action of lncRNA147410.3 in T. gondii infected mouse brain, providing a scientific basis for using lncRNA147410.3 as a therapeutic target to treat neurological disorder induced by T. gondii.

Downregulation of lncRNA-11496 in the Brain Contributes to Microglia Apoptosis via Regulation of Mef2c in Chronic T. gondii Infection Mice.

Though it is well known that chronic infections of Toxoplasma gondii (T. gondii) can induce mental and behavioral disorders in the host, little is known about the role of long non-coding RNAs (lncRNAs) in this pathological process. In this study, we employed an advanced lncRNAs and mRNAs integration chip (Affymetrix HTA 2.0) to detect the expression of both lncRNAs and mRNAs in T. gondii Chinese 1 strain infected mouse brain. As a result, for the first time, the downregulation of lncRNA-11496 (NONMMUGO11496) was identified as the responsible factor for this pathological process. We showed that dysregulation of lncRNA-11496 affected proliferation, differentiation and apoptosis of mouse microglia. Furthermore, we proved that Mef2c (Myocyte-specific enhancer factor 2C), a member of the MEF2 subfamily, is the target gene of lncRNA-11496. In a more detailed study, we confirmed that lncRNA-11496 positively regulated the expression of Mef2c by binding to histone deacetylase 2 (HDAC2). Importantly, Mef2c itself could coordinate neuronal differentiation, survival, as well as synapse formation. Thus, our current study provides the first evidence in terms of the modulatory action of lncRNAs in chronic toxoplasmosis in T. gondii infected mouse brain, providing a solid scientific basis for using lncRNA-11496 as a therapeutic target to treat T. gondii induced neurological disorder.

miRNA and circRNA expression patterns in mouse brain during toxoplasmosis development.

BACKGROUND: Increasing evidence has shown that circular RNAs (circRNAs) are involved in neurodegenerative disorders, but their roles in neurological toxoplasmosis are yet to know. This study examined miRNA and circRNA expressions in mouse brain following oral infection with T. gondii Pru strain. RESULTS: Total RNA extracted from acutely infected (11 days post infection (DPI)), chronically infected (35 DPI) and uninfected mouse brain samples were subjected to genome-wide small RNA sequencing. In the acutely infected mice, 9 circRNAs and 20 miRNAs were upregulated, whereas 67 circRNAs and 28 miRNAs were downregulated. In the chronically infected mice, 2 circRNAs and 42 miRNAs were upregulated, whereas 1 circRNA and 29 miRNAs were downregulated. Gene ontology analysis predicted that the host genes that produced the dysregulated circRNAs in the acutely infected brain were primarily involved in response to stimulus and ion binding activities. Furthermore, predictive interaction networks of circRNA-miRNA and miRNA-mRNA were constructed based on genome-wide transcriptome sequencing and computational analyses, which might suggest the putative functions of miRNAs and circRNAs as a large class of post-transcriptional regulators. CONCLUSIONS: These findings will shed light on circRNA-miRNA interactions during the pathogenesis of toxoplasmosis, and they will lay solid foundation for studying the potential regulation roles of miRNAs and circRNAs in T. gondii induced pathogenesis.

Molecular Detection of Toxoplasma gondii in the Slaughter Sheep and Goats from Shandong Province, Eastern China.

It is generally recognized that sheep are susceptible to Toxoplasma gondii and play a very important role in the transmission of toxoplasmosis to humans. In China, sheep toxoplasmosis has been reported in some regions based on serological investigations. However, little is known about sheep toxoplasmosis in Shandong province, eastern China. Thus, this study was conducted to investigate the prevalence of T. gondii infection in the slaughter sheep and goats from three cities (Weihai, Yantai, and Rizhao) of Shandong province, eastern China. From November 2016 to March 2018, a total of 692 meat samples (438 sheep and 254 goats) were collected and detected by a seminested PCR-targeted T. gondii B1 gene. The overall prevalence of T. gondii in sheep and goats were 9.84% and 10.73%, respectively. Meat collected from rural markets (16.04%) had a significantly higher T. gondii prevalence than those collected from supermarkets (6.84%) (p < 0.001). Moreover, sheep and goats raised in backyard were more easily to be infected by T. gondii compared with those raised in farms (p < 0.001). This is the first report of the molecular prevalence of T. gondii infection in sheep and goats in Shandong province, eastern China, which would provide effective data for prevention and control of sheep and human toxoplasmosis in China.

L-Tryptophan represses persister formation via inhibiting bacterial motility and promoting antibiotics absorption.

Aim: The bacterial persisters have emerged as a huge threat to human health. Here, we investigated the role of L-tryptophan in bacterial persister killing by aminoglycoside antibiotics (AGs). Materials & methods: The relevance to the antibiotic susceptibility of Escherichia coli including transcriptional sequencing, gene expression, intracellular ATP, Nicotinamide adenine dinucleotide (NAD/NADH), reactive oxygen species and membrane depolarization were determined. Results & conclusion: We found that exogenous L-tryptophan efficiently inhibited AGs-enabled persisters. The flagellar genes were almost significantly downregulated. Besides, the AGs uptake was obviously increased as the result of elevation in proton motive force (PMF) in response to L-tryptophan-mediated NADH production. Taken together, these data supported a novel role of L-tryptophan in eradicating AGs persisters against E. coli.

Immunogenicity of a Virus-Like-Particle Vaccine Containing Multiple Antigenic Epitopes of Toxoplasma gondii Against Acute and Chronic Toxoplasmosis in Mice.

There is no effective protective vaccine against human toxoplasmosis, which is a potential threat to nearly a third of the world population. Vaccines based on virus-like particles (VLPs) have been highly successful in humans for many years, but have rarely been applied against Toxoplasma gondii infection. In this study, we inserted a B cell epitope (SAG182-102 or SAG1301-320), a CD8+ cell epitope (HF10 or ROP7), and a CD4+ cell epitope (AS15) of T. gondii into a truncated HBcΔ(amino acids1-149) particle to construct four chimeric VLP vaccine formulations, i.e., HBcΔH82, HBcΔH301, HBcΔ R82, and HBcΔ R301. When these chimeric HBc particles were expressed in Escherichia coli, they showed icosahedral morphology similar to that of the original VLPs and were evaluated as vaccine formulations against acute and chronic toxoplasmosis in a mouse model (BALB/c mice (H-2d). All these chimeric HBc VLPs induced strong humoral and cellular immune responses with high IgG antibody titers and interferon(IFN)-γ production. Only the mice immunized with HBcΔH82 showed prolonged survival time (15.6 ± 3.8 vs. 5.6 ± 0.8 days) against acute infection with RH tachyzoites and decrease in brain parasite load (1,454 ± 239 vs. 2,091 ± 263) against chronic infection with Prugniuad cysts, as compared to the findings for the control group. These findings suggest that HBc VLPs would act as an effective carrier for delivering effective multiple antigenic epitopes and would be beneficial for developing a safe and long-acting vaccine against toxoplasmosis.

From inflammatory reactions to neurotransmitter changes: Implications for understanding the neurobehavioral changes in mice chronically infected with Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite that can cause a latent infection in the central nervous system, leading to neurobehavioral abnormalities in the host. However, the mechanism underlying these changes remains relatively unexplored. In this study, we detected behavioral changes, pathological injury, secretion of neurotransmitters and related signal pathway in mice infected by T. gondii using behavioral test, histopathology, immunofluorescence staining, western blotting, HPLC and real time PCR. Mice showed neurobehavioral disturbances two months after infection with T. gondii. Histopathology revealed the activation of astrocytes and microglia, apoptosis of neurons and decreases in synapses in the brain of infected mice. Excessive secretion of cytokines and chemokines was detected in the brains of mice infected by T. gondii compared to uninfected mice. Furthermore, T. gondii infection led to abnormalities in neurotransmitters and the activation of NF-κB and dopamine (DA) signaling pathways in the infected mice. In conclusion, excessive activation of the inflammation in the brain could induce neuronal apoptosis in mice chronically infected with T. gondii. Dysregulation of the dopaminergic neurotransmitter could provide an explanation of neurobehavioral disorders in infected hosts.

Knowledge of influenza among primary and secondary school teachers in Huainan City / 中国学校卫生

Objective@#To understand knowledge and practice of influenza prevention and associated factors among primary and middle school teachers, and to provide a reference for conducting the relevant propaganda work of influenza.@*Methods@#Stratified random cluster sampling method was used to select 858 primary and secondary school teachers from Huainan of Anhui Province to complete questionnaires on influenza prevention.@*Results@#Television and radio broadcasting were the basic tools for teachers to acquire flu knowledge, with 74.12% and 80.00% of primary and secondary school teachers, respectively. The total awareness rate of influenza knowledge among primary and middle school teachers was 56.63% and 58.63%, respectively. The results of univariate analysis showed primary and secondary school teachers’ influenza awareness were significantly affected by regions and education levels. In addition, primary school teachers’ influenza awareness was significantly affected by full-time medical technicians or part-time medical workers, secondary school teachers’ influenza awareness was significantly affected by working years(P<0.05). The total formation rates of influenza-related health behavior between primary and secondary school teachers were 71.20% and 73.00%, respectively. The results of univariate analysis showed primary and secondary school teachers’ influenza health behaviors was significantly affected by regions, educational levels, full-time medical technicians or part-time medical workers and health training. Moreover, secondary school teachers’ influenza health behaviors was significantly affected by working years(P<0.05). Multivariate logistic regression analysis showed that whether the comprehensive scores of influenza knowledge and behavior were qualified was correlation with the regional and educational levels. Additionally, the factors whether it is a full-time medical technician or a part-time medical teacher also had an effect on the comprehensive score of behavior(P<0.05).@*Conclusion@#The influenza knowledge level and the health behavior formation rate in the primary and secondary school teachers need to be improved, the knowledge of influenza and the relevant influencing factors should be taken into consideration to take targeted health intervention measures to improve their ability to fight against influenza.

Chitosan Microsphere Used as an Effective System to Deliver a Linked Antigenic Peptides Vaccine Protect Mice Against Acute and Chronic Toxoplasmosis.

Multiple antigenic peptide (MAP) vaccines have advantages over traditional Toxoplasma gondii vaccines, but are more susceptible to enzymatic degradation. As an effective delivery system, chitosan microspheres (CS) can overcome this obstacle and act as a natural adjuvant to promote T helper 1 (Th1) cellular immune responses. In this study, we use chitosan microparticles to deliver multiple antigenic epitopes from GRA10 (G10E), containing three dominant epitopes. When G10E was entrapped within chitosan microparticles (G10E-CS), adequate peptides for eliciting immune response were loaded in the microsphere core and this complex released G10E peptides stably. The efficiency of G10E-CS was detected both in vitro, via cell culture, and through in vivo mouse immunization. In vitro, G10E-CS activated Dendritic Cells (DC) and T lymphocytes by upregulating the secretion of costimulatory molecules (CD40 and CD86). In vivo, Th1 biased cellular and humoral immune responses were activated in mice vaccinated with G10E-CS, accompanied by significantly increased production of IFN-γ, IL-2, and IgG, and decreases in IL-4, IL-10, and IgG1. Immunization with G10E-CS conferred significant protection with prolonged survival in mice model of acute toxoplasmosis and statistically significant decreases in cyst burden in murine chronic toxoplasmosis. The results from this study indicate that chitosan microspheres used as an effective system to deliver a linked antigenic peptides is a promising strategy for the development of efficient vaccine against T. gondii.

Structural predication and antigenic analysis of Toxoplasma gondii ROP20.

Toxoplasma gondii infects almost all the warm-blooded animals. ROP20 protein is expressed in the rhoptry of Toxoplasma gondii. In this study, the secondary structure of ROP20 was analyzed using SMART software. We constructed and analyzed the 3D model of ROP20 protein using SWISS-MODEL online procedure and Visual Molecular Dynamics (VMD) software. The structure analysis fully indicated that ROP20 protein is an important member of the ROP family. Furthermore, We used DNASTAR software and Epitope Database online service to analyze liner-B cell epitopes and T-cell epitopes of ROP20 protein. All the analysis results of ROP20 protein can provide positive information on treatment and vaccine for toxoplasmosis. Moreover, ROP20 gene was obtained from PCR, and a recombinant eukaryotic expression vector (pEGFP-C1-ROP20) was constructed in the following study. After restriction enzyme digestion, the constructed plasmid was transfected into HEK 293-T cells. The RT-PCR result indicated that the recombinant plasmid could transcribe successfully in HEK 293-T cell. The results of western blotting indicated the expressed proteins can be recognized by anti-STAg mouse sera.

Exosomes derived from Toxoplasma gondii stimulate an inflammatory response through JNK signaling pathway.

AIM: Exosomes are nanoscale membranous vesicles secreted by most cell types able to transfer bioactive molecules among cells, which play crucial roles in intercellular communication. We characterized the exosomes derived from Toxoplasma gondii and detected the immune response in macrophages. METHODS: We used transmission electron microscopy, nanotracking analysis and western blotting to identify T. gondii exosomes. Functional experiments were performed in RAW264.7 cells for the induction of cytokines, MAPKs (p38 MAPK, ERK 1/2 and c-Jun N-terminal kinase [JNK]), mRNAs and nuclear translocation of phosphorylated JNK protein. RESULTS: JNK pathway was activated by T. gondii exosomes, and the production of IL-12, IFN-γ and TNF-α was significantly increased in macrophages. CONCLUSION: Our findings demonstrated that T. gondii exosomes elicit innate immune through JNK activation, which could provide new insight into the essential regulators of host-pathogen interactions.

Characterization of exosomes derived from Toxoplasma gondii and their functions in modulating immune responses.

INTRODUCTION: Exosomes are nanograde membrane-bound vesicles secreted from most cell types through the fusion of multivesicular bodies with plasma membranes. Some of these exosomes are well defined, and are known to have immunomodulatory properties as well as play critical roles in intercellular communications. In this study, we characterized the exosomes derived from Toxoplasma gondii and their functions in aspect of immune responses. METHODS: T. gondii exosomes were isolated and identified using electron microscopy, nanoparticle tracking analysis, and Western blotting. The viability of macrophage RAW264.7 cells affected by exosomes was evaluated using a Cell Counting Kit (CCK-8). Then the uptake of T. gondii exosomes by RAW264.7 cells was detected by labeling with fluorescent dye PKH67. After exosomes stimulation, in vitro the production of interleukin (IL)-12, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10 in RAW264.7 cells were investigated using enzyme-linked immunosorbent assay (ELISA). In immunized BALB/c mice, the antibodies, cytokines as well as the percentage of CD4+ and CD8+ T cells were determined using ELISA and flow cytometric analysis. Protective efficacy was evaluated by challenging intraperitoneally with tachyzoites of T. gondii. RESULTS: We successfully isolated and characterized the exosomes derived from T. gondii. Functionally, the viability of macrophage RAW264.7 cells was significantly affected by exosomes at a high concentration (160 µg/mL). The production of IL-12, TNF-α and IFN-γ in macrophage cells were increased, and the level of IL-10 was decreased. Furthermore, BALB/c mice immunized with T. gondii exosomes showed both humoral and cellular immune responses and also exhibited a prolonged survival time. CONCLUSION: T. gondii exosomes could modulate macrophage activation in vitro and trigger humoral and cellular immune responses and partial protection against acute parasite infection in mice, which suggested that exosomes may serve as a potential candidate against toxoplasmosis.

DNA Vaccines Encoding Toxoplasma gondii Cathepsin C 1 Induce Protection against Toxoplasmosis in Mice.

Toxoplasma gondii cathepsin C proteases (TgCPC1, 2, and 3) are important for the growth and survival of T. gondii. In the present study, B-cell and T-cell epitopes of TgCPC1 were predicted using DNAstar and the Immune Epitope Database. A TgCPC1 DNA vaccine was constructed, and its ability to induce protective immune responses against toxoplasmosis in BALB/c mice was evaluated in the presence or absence of the adjuvant α-GalCer. As results, TgCPC1 DNA vaccine with or without adjuvant α-GalCer showed higher levels of IgG and IgG2a in the serum, as well as IL-2 and IFN-γ in the spleen compared to controls (PBS, pEGFP-C1, and α-Galcer). Upon challenge infection with tachyzoites of T. gondii (RH), pCPC1/α-Galcer immunized mice showed the longest survival among all the groups. Mice vaccinated with DNA vaccine without adjuvant (pCPC1) showed better protective immunity compared to other controls (PBS, pEGFP-C1, and α-Galcer). These results indicate that a DNA vaccine encoding TgCPC1 is a potential vaccine candidate against toxoplasmosis.

Structuraland antigenic analysis of a new Rhoptry Pseudokinase Gene (ROP54) in Toxoplasma gondii.

Toxoplasma gondii is defined as an obligate intracellular apicomplexan parasite and influences approximatelyone-third of the human all over the world. ROP54 protein is expressed in the rhoptry of Toxoplasma gondii. In the present study, we used SMART software to analyzethe secondary structure of ROP54. The 3D model of ROP54 protein was constructed and analyzed using SWISS-MODEL server and VMD software. The structure results fully showed that ROP54 proteinis an importantmember from the ROP family. Moreover, DNAMAN software and Epitope Database online service were used to analyze liner-B cell epitopes and Th-cell epitopes of the protein. The bioinformatics prediction of ROP54 protein could provide positive information on treatment and vaccine for toxoplasmosis. Furthermore, ROP54 gene was obtained from PCR, and a recombinant eukaryotic expression vector (pEGFP-ROP54) was constructed in the following study. After identification of enzyme digestion, the constructed plasmid was transfected into HEK 293-T cells. The RT-PCR result suggested that the recombinant plasmid could transcribe successfully in HEK 293-T cell.

SAG5B and SAG5C combined vaccine protects mice against Toxoplasma gondii infection.

Infections with the protozoan parasite Toxoplasma gondii, which are common around the world, can lead to congenital infections in humans. T. gondii surface antigen protein 5B (SAG5B) and SAG5C are potential stimulators of humoral and cellular immune responses. In this study, a multi-antigenic DNA vaccine constructed to express T. gondii SAG5B and SAG5C proteins simultaneously was used to immunize BALB/c mice to evaluate the protective efficacy of the vaccine. IgG antibody and gamma interferon (IFN-γ) cytokine production in the pSAG5B/SAG5C DNA vaccine group were significantly higher (0.853±0.103 and 915.2±106.9, respectively) than in the single DNA vaccine groups (pSAG5B, 0.667±0.109 and 598.3±74.9, respectively; pSAG5C, 0.696±0.092 and 623.7±95.5, respectively). After a lethal challenge with 1×104 RH strain tachyzoites, the survival time of the mice (17days) immunized with pSAG5B/SAG5C was longer than that of the single-gene-immunized mice (12days) or the control mice (6days). Moreover, after intragastric infection with 20 T. gondii PRU (low virulence) strain cysts, the number of brain cysts in the pSAG5B/SAG5C-vaccinated mice was only 25% of the number for the PBS-injected mice. Our findings indicate that, in comparison with the other mouse groups, the multi-antigenic DNA vaccine (pSAG5B/SAG5C) significantly induced immune responses and improved the protection against challenge with T. gondii in the host animals.

Immunization with a DNA vaccine encoding Toxoplasma gondii Superoxide dismutase (TgSOD) induces partial immune protection against acute toxoplasmosis in BALB/c mice.

BACKGROUND: Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects all warm-blooded animals including humans and causes toxoplasmosis. An effective vaccine could be an ideal choice for preventing and controlling toxoplasmosis. T. gondii Superoxide dismutase (TgSOD) might participate in affecting the intracellular growth of both bradyzoite and tachyzoite forms. In the present study, the TgSOD gene was used to construct a DNA vaccine (pEGFP-SOD). METHODS: TgSOD gene was amplified and inserted into eukaryotic vector pEGFP-C1 and formed the DNA vaccine pEGFP-SOD. Then the BALB/c mice were immunized intramuscularly with the DNA vaccine and those injected with pEGFP-C1, PBS or nothing were treated as controls. Four weeks after the last immunization, all mouse groups followed by challenging intraperitoneally with tachyzoites of T. gondii ME49 strain. RESULTS: Results showed higher levels of total IgG, IgG2α in the sera and interferon gamma (IFN-γ) in the splenocytes from pEGFP-SOD inoculated mice than those unvaccinated, or inoculated with either empty plasmid vector or PBS. The proportions of CD4+ T cells and CD8+ T cells in the spleen from pEGFP-SOD inoculated mice were significantly (p < 0.05) increased compared to control groups. In addition, the survival time of mice immunized with pEGFP-SOD was significantly prolonged as compared to the controls (p < 0.05) although all the mice died. CONCLUSION: The present study revealed that the DNA vaccine triggered strong humoral and cellular immune responses, and aroused partial protective immunity against acute T. gondii infection in BALB/c mice. The collective data suggests the SOD may be a potential vaccine candidate for further development.

Protection via a ROM4 DNA vaccine and peptide against Toxoplasma gondii in BALB/c mice.

BACKGROUND: Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite with a broad host range including most warm-blooded animals, including humans. T. gondii surface antigen 1 (SAG1) is a well-characterized T. gondii antigen. T. gondii expresses five nonmitochondrial rhomboid intramembrane proteases, TgROM1-5. TgROM4 is uniformly distributed on the surface of T. gondii and involved in regulating MIC2, MIC3, MIC6, and AMA1 during T. gondii invasion of host cells. Bioinformatics have predicted ROM4 B-cell and T-cell epitopes. Immunization strategy is also a key factor in determining the effectiveness of the immune response and has gained increasing attention in T. gondii vaccine research. In this study, we used a DNA prime-peptide boost vaccination regimen to assess the protective efficacy of various vaccination strategies using TgROM4. METHODS: We identified a polypeptide (YALLGALIPYCVEYWKSIPR) using a bioinformatics approach, and immunized mice using a DNA-prime and polypeptide-boost regimen. BALB/c mice were randomly divided into six groups, including three experimental groups (peptide, pROM4 and pROM4/peptide) and three control groups (PBS, pEGFP-C1 and pSAG1). Mice were then immunized intramuscularly four times. After immunization, IgG and cytokine productions were determined using enzyme-linked immunosorbent assays. The survival time of mice was evaluated after challenge with tachyzoites of T. gondii RH strain. Additionally, the number of cysts in the brain was determined after intragastric challenge with cysts of T. gondii PRU strain. RESULTS: Mice vaccinated with different immunization regimens (peptide, pROM4 and pROM4/peptide) elicited specific humoral and cellular responses, with high levels of IgG, IgG2a, and interferon (IFN)-γ. Moreover, IgG, IgG2a and IFN-γ levels were highest in the pROM4/peptide group. Immunized mice, especially those in the pROM4/peptide group, had prolonged survival times after challenge with tachyzoites and reduced numbers of brain cysts after infection compared with negative controls. CONCLUSION: A DNA prime-peptide boost regimen based on ROM4 elicited the highest level of humoral and cellular immune responses among immunization regimens, and may be a promising approach to increase the efficacy of DNA immunization.

Vaccination with toxofilin DNA in combination with an alum-monophosphoryl lipid A mixed adjuvant induces significant protective immunity against Toxoplasma gondii.

BACKGROUND: A widely prevalent disease, toxoplasmosis poses serious health threats to both humans and animals; therefore, development of an ideal DNA vaccine against Toxoplasma gondii is needed eagerly. The purpose of the present study is to assess the protective efficacy of a DNA vaccine encoding the T. gondii toxofilin gene (pEGFP-toxofilin). In addition, toxofilin DNA vaccine combined with the individual adjuvants, alum or monophosphoryl lipid A (MPLA), or a mixture of alum-MPLA adjuvant were screened for their ability to enhance antibody responses. METHODS: Using bioinformatics, we analyzed the gene and amino acid sequences of the toxofilin protein, recognizing and identifying several potential linear B and T helper (Th)-1 cell epitopes. BALB/c mice were immunized three times with either toxofilin DNA vaccine alone or in combination with the adjuvants such as alum, MPLA or an alum-MPLA mixture. The systemic immune response was evaluated by cytokine, the percentage of CD4 (+) and CD8 (+) T cells and specific antibody measurement. Two weeks after the last immunization, protective efficacy was evaluated by challenging intraperitoneally with 1 × 104 tachyzoites of T. gondii or intragastrically with 20 cysts of T. gondii PRU strain. RESULTS: All experimentally immunized mice developed strong humoral and cellular immune responses compared with the control groups. Moreover, by comparison with the non-adjuvant toxofilin DNA vaccine group, adding alum adjuvant to toxofilin DNA vaccine resulted in an increase in humoral response and a skewed Th2 response. However, the MPLA adjuvant with toxofilin DNA vaccine induced significantly enhanced humoral and Th1-biased immune responses. Importantly, the co-administration of alum-MPLA adjuvant in combination with the toxofilin DNA vaccine shifted the Th2 immune response to a Th1 response compared with the alum-toxofilin group, and elicited the strongest humoral and Th1 responses among all the groups. At the same time, a longer survival time and less cyst amounts against T. gondii infection were also observed in the alum-MPLA-toxofilin group in comparison with single or no adjuvant groups. CONCLUSIONS: Toxoplasma gondii toxofilin is a promising vaccine candidate that warrants further development. Co-administration of the alum-MPLA adjuvant mixture with DNA vaccine could effectively enhance immunogenicity and strongly skew the cellular immune response towards a Th1 phenotype.

Serum Metabolic Profiling of Oocyst-Induced Toxoplasma gondii Acute and Chronic Infections in Mice Using Mass-Spectrometry.

Toxoplasma gondii is an obligate intracellular parasite causing severe diseases in immunocompromised individuals and congenitally infected neonates, such as encephalitis and chorioretinitis. This study aimed to determine whether serum metabolic profiling can (i) identify metabolites associated with oocyst-induced T. gondii infection and (ii) detect systemic metabolic differences between T. gondii-infected mice and controls. We performed the first global metabolomics analysis of mice serum challenged with 100 sporulated T. gondii Pru oocysts (Genotype II). Sera from acutely infected mice (11 days post-infection, dpi), chronically infected mice (33 dpi) and control mice were collected and analyzed using LC-MS/MS platform. Following False Discovery Rate filtering, we identified 3871 and 2825 ions in ESI+ or ESI- mode, respectively. Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) identified metabolomic profiles that clearly differentiated T. gondii-infected and -uninfected serum samples. Acute infection significantly influenced the serum metabolome. Our results identified common and uniquely perturbed metabolites and pathways. Acutely infected mice showed perturbations in metabolites associated with glycerophospholipid metabolism, biosynthesis of amino acid, and tyrosine metabolism. These findings demonstrated that acute T. gondii infection induces a global perturbation of mice serum metabolome, providing new insights into the mechanisms underlying systemic metabolic changes during early stage of T. gondii infection.