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.

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.

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.

[Construction and expression of an eukaryote vector of 14-3-3 protein in Toxoplasma gondii].

OBJECTIVE: To construct and express the eukaryotic expression vector of 14-3-3 protein of Toxoplasma gondii RH stain. METHODS: The structure and physicochemical property of 14-3-3 protein were predicted by bioinformatics analysis tools. The desired gene fragment was amplified from total RNA in T. gondii RH strain by RT-PCR, and sub-cloned into pcDNA3.0 to construct recombinant plasmid pcDNA3.0/14-3-3. After PCR confirming, double restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector pcDNA3.0/14-3-3 was transfected into HeLa cells and the target protein was detected by Western blotting. RESULTS: The prediction of its gene sequence and amino acid sequence suggested that the 14-3-3 protein was acid soluble protein with five conserved regions, existing as homo- or hetero-dimers. The amplified gene fragment was about 800 bp, and the inserted fragment in pcDNA3.0/14-3-3 was 801 bp by sequencing, which had 99% homology to the 14-3-3 gene sequence of T. gondii in GenBank (Accession No. AB012775.1). Western blotting showed that there was more 14-3-3 protein expressed in the pcDNA3.0/14-3-3-transfected HeLa cells than not-transfected and mock transfected cells. Its relative molecular mass (Air) was about 30 000. CONCLUSION: The eukaryotic expression vector pcDNA3.0/14-3-3 is constructed and expressed in eukaryotic cells.

Protective immunity induced in mice by multiantigenic DNA vaccine with genes encoding SAG1 and MIC8 of Toxoplasma gondii.

OBJECTIVE: To observe the immunoprotection induced by multiantigenic SAG1-MIC8 DNA vaccine of Toxoplasma gondii in C57BL/6J mice. METHODS: The sequences of genes encoding SAG1 and MIC8 protein were inserted into the eukaryotic expression vector pcDNA3.1 and the multiantigenic recombinant plasmid pcDNA3.1-SAG1-MIC8 was constructed. Then the recombinant plasmid was transfected into Hela cells to test its expression and the recombinant protein characterized by Western blotting 70 mice were divided into 5 groups randomly: PBS, pcDNA3.1, pcDNA3.1-SAG1, pcDNA3.1-MIC8 and pcDNA3.1-SAG1-MIC8. Each mouse was injected intra-muscularly by 100 microg recombinant plasmid for 3 times every two weeks. Mice were bled on day 0, 13, 27, 41, and 55. Four weeks after the final inoculation (on day 56), spleens from seven immunized mice per group were collected. Another seven immunized mice per group were intraperitoneally challenged with 1 x 10(4) tachyzoites of RH T. gondii and the survival time was observed. Serum IgG antibody and cytokines IFN-gamma and IL-4 were demonstrated by ELISA and the T lymphocyte proliferation assay were carried out with 3H-TdR incorporation. RESULTS: Western blotting showed that the mature protein extracts in Hela cells upon transfection with pcDNA3.1-SAG1 (Mr 34,000), pcDNA3.1-MIC8 (Mr 74,000) and pcDNA3.1-SAG1-MIC8 (Mr 109,000) were effectively expressed in cells. The results of IgG antibodies (on day 41 and 55), IgG2b, IgG2c, IFN-gamma (on day 55) and T lymphocyte proliferation assay (on day 56) were more obvious in mice immunized with pcDNA3.1-SAG1-MIC8 multiantigenic DNA vaccine than those in mice with single-gene plasmids (P < 0.05). There was no significant difference in IgG1 and IL-4 levels between vaccinated and control mice after the final inoculation (on day 55) (P > 0.05). The median survival time was 3, 4, 7, 7, and 10d, respectively, with considerable difference among the groups (P < 0.01). CONCLUSION: The multiantigenic DNA vaccine elicits a stronger immuno-protection in mice than the monovalent DNA vaccine.

[Effect of Escherichia coli heat-labile enterotoxin B subunit on SAG1-ROP2 compound gene vaccine of Toxoplasma gondii tachyzoite].

OBJECTIVE: To investigate the effect of Escherichia coil heat-labile enterotoxin B subunit (LTB) as a genetic adjuvant in enhancing the immune response induced by Toxoplasma gondii tachyzoite compound gene vaccine. METHODS: The eukaryotic expression plasmids of pcDNA3.1-SAG1-ROP2 and pEASY-E1-LTB were constructed. Eighty-eight BALB/c mice were randomly divided into four groups: PBS (group A), pcDNA3.1(B), pcDNA3.1-SAG1-ROP2 (C) and pcDNA3.1-SAG1-ROP2+pEASY-E1-LTB (D). Fifteen mice in each group were randomly selected, and intranasally immunized weekly with 20 microg plasmid or 20 microl PBS, respectively. Each mouse received four immunizations with the same dose of antigen. Two weeks after the final immunization, the antibodies and cytokines were detected, including the specific IgG and IgA antibodies in serum, sIgA in mucosa douche, IFN-gamma and IL-4 in splenocyte culture supernatant. The remaining mice in each group were immunized three times weekly with 20 microg plasmid or 20 microl PBS, respectively, and challenged by T. gondii tachyzoites at four weeks after the final vaccination (1 x 10(3) per mouse). The survival time of mice was recorded. RESULTS: The recombinant plasmids pEASY-E1-LTB were constructed. The specific IgG (0.626/- 0.100) and IgA antibodies (1.086 +/- 0.138) in serum, sIgA (0.886 +/- 0.164) in mucosa douche, cytokines IFN-gamma [(2017 +/- 266) pg/ml] and IL-4 [(203.31) pg/ml] in splenocyte culture supernatant in group D were all higher than those in other groups (P < 0.05). After challenged with T. gondii tachyzoites, the median survival time of mice in groups A, B, C, and D were 3, 4, 6, and 10 d, respectively. The survival time of mice in group D was longest (P < 0.05). CONCLUSION: E. coil heat-labile enterotoxin can enhance the immune response induced by the compound gene vaccine of T. gondii tachyzoites.

[Oral mixed DNA vaccine protects mice from infection of Toxoplasma gondii].

OBJECTIVE: To examine the immune response in BALB/c mice induced by oral mixed Toxoplasma gondii DNA vaccine delivered by live attenuated Salmonella typhimurium. METHODS: Gene fragments SAG1 and SAG2 were amplified from the genomic DNA of T. gondii RH strain by PCR and were subcloned into pcDNA3.1(+/-) eukaryotic expression vector. The positive recombinant plasmid was transformed into aroA- and aroD-attenuated Salmonella typhimurium BRD509 (BRD509/pSAG1/SAG2). After screened by PCR, restrictive enzyme digestion and sequencing, recombinant Salmonella strain was used to immunize BALB/c mice by i.g. route, three times at 2-week interval. Humoral and cellular responses were assayed using ELISA for determining antibody, IFN-gamma and IL-4. MTT assay was used to detect the proliferation activity of T lymphocytes and the activity of NK killers. FCM was also used to sort the lymphocyte subsets of spleen cells All mice were then infected with highly virulent RH tachyzoites of T. gondii intraperitoneally. RESULTS: Significant increase of specific IgG level was observed in immunized mice with a titer of 1:100. Proliferation activity of specific NK cells and T lymphocytes was highly enhanced in BRD509/ pSAG1/SAG2 vaccinated mice: the killing activity of NK cells was 85% +/- 7%, the proliferation SI of T lymphocytes was 2.83, which resulted in a 5 days longer survival time than mice in control group after challenge infection. CONCLUSION: The oral mixed DNA vaccine delivered by attenuated Salmonella typhimurium shows an immunoprotection against T. gondii in mice.

[Construction of monovalent and compound nucleic acid vaccines against Toxoplasma gondii with gene encoding p30].

OBJECTIVE: To construct a monovalent gene vaccine pcDNA3.1-p30 and a compound gene vaccine pcDNA3.1-p30-ROP2 and assess the protective effect of the two vaccines against Toxoplasma gondii. METHODS: The sequences encoding p30 and ROP2 were amplified from the genomic DNA of T. gondii RH strain by polymerase chain reaction (PCR) and inserted into eukaryotic vector pcDNA3.1 to construct pcDNA3.1-p30 and pcDNA3.1-p30-ROP2. Mice were injected with the recombinant plasmid to observe the immunoprotectivity of the nucleic acid vaccine by using ELISA for detection of total IgG and observing the survival time after tachyzoites challenge. RESULTS: The recombinant plasmids pcDNA3.1-p30 and pcDNA3.1-p30-ROP2 were constructed. Mice in pcDNA3.1-p30-ROP2 group showed higher IgG (P < 0.05) and survived longer than those in pcDNA3.1-p30 group (P < 0.01) after challenged with T. gondii. CONCLUSION: Compound vaccine of genes from different stages of T. gondii elicits stronger immunoprotectivity in mice than a single gene vaccine.