XinChao Liu

Immunological changes induced by Toxoplasma gondii Glutathione-S-Transferase (TgGST) delivered as a DNA vaccine.

In this study, a DNA vaccine (pTgGST) encoding T. gondii antioxidant glutathione-S-transferase (TgGST) inserted into eukaryotic expression vector pVAX I was constructed and the immune protective efficacy of intramuscular vaccination of mice with pTgGST was analyzed. Mice immunized with pTgGST elicited high titers of total IgG, IgG1, IgG2a, IgA and IgM antibodies, while IgE showed no changes. Also, significant cytokine production of IFN-γ, IL-4 and IL-17 was detected in mice immunized with pTgGST, but not TGF-β1. CD8(+) T cells subsets and MHC-I molecules showed significant increase in contrast to CD4(+) subsets. Immunization with pTgGST significantly prolonged survival time (14 days) after challenge infection with the virulent T. gondii RH strain, compared with the control groups which died within 8 days. These results suggested that TgGST DNA vaccine could trigger strong humoral and cellular responses and induce partial protection against acute toxoplasmosis.

Pathogenicity of two Toxoplasma gondii strains in chickens of different ages infected via intraperitoneal injection

This experiment was conducted to investigate the pathogenicity of Toxoplasma gondii in broilers of different ages. Chickens at the ages of 7, 14, 21 and 28 days were injected intraperitoneally with 1 × 108 tachyzoites of RH and JS strains of T. gondii, respectively. The clinical signs and death of chickens were recorded daily post inoculation. Serum samples were collected at days 0, 4, 11, 18, 25, 32, 39, 46 and 53 post infection to screen T. gondii circulating antigens (TCA) and T. gondii circulating antibodies (TCAb). The results showed that T. gondii infection of 7-day-old chickens caused death, even though the mortality rate of the JS strain (100%) was significantly higher than that of the RH strain (70%). Chickens at 14 days old showed only mild clinical signs, but no death. Neither clinical signs nor death were recorded in 21-day-old and 28-day-old chickens. TCA and TCAb became positive at days 4 and 11, respectively. Both the TCA and the TCAb of groups 21 days old (RH strain) and 28 days old (both RH and JS strains) decreased to a negative level earlier than the other experimental groups. Specific T. gondii DNA was detected by polymerase chain reaction in chickens that survived in the 7-day-old group (RH strain) and in all infected chickens of groups 14 days old and 21 days old injected with both strains. In the groups injected at 28 days old, three samples (RH strain) and one sample (JS strain) were found negative. The results indicated that the age of the chicken was an important factor affecting the pathogenicity of T. gondii and that these two strains of T. gondii displayed different virulence for chickens.

The Molecular Characterization and Immunity Identification of Microneme 3 of Eimeria acervulina.

The gene of Eimeria acervulina microneme protein 3 (EaMIC3) was cloned and characterized. According to the conserved sequence, the 3′‐ and 5′‐ends of EaMIC3 were amplified by the rapid amplification of cDNA ends (RACE). The full length cDNA of this gene was obtained by overlapping the sequences of 3′‐ and 5′‐extremities and amplification by reverse transcription PCR. The sequence analysis revealed that the opening reading frame (ORF) of EaMIC3 was 2,607 bp and encoded a protein of 868 amino acids with 93.04 kDa. Western blotting assay showed that the recombinant protein was successfully recognized by the sera of chickens experimentally infected with E. acervulina, whereas the native protein in the somatic extract of sporozoites was as well detected by sera from rats immunized with the recombinant protein of EaMIC3. Immunofluorescence analysis indicated that EaMIC3 was expressed in the sporozoites and merozoites stages of E. acervulina. Animal challenge experiments demonstrated that the recombinant protein of EaMIC3 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented anticoccidial index (ACI) more than 165. Moreover, EaMIC3 protein produced significantly high level of IgG antibody, IFN‐γ, IL‐10, IL‐17 TGF‐β, CD4+, and CD8+.

Detection of Toxoplasma gondii in chicken and soil of chicken farms in Nanjing region, China

BackgroundSoil is increasingly recognized as an important source in the transmission of Toxoplasma gondii (T. gondii). The aim of this study was to investigate the presence of T. gondii in the soil and to grasp the relationships between the contamination of soil and chicken infections.MethodsPCR method based on T. gondii-conserved gene internal transcribed spacer 1 (ITS-1) as target gene and ELISA method (sGRA8-ELISA) using the recombinant protein of shortened GRA8 gene of T. gondii as antigen were developed and applied. From April 2013 to March 2014, a total of 700 soil samples were collected at various sites located in thirty farms categorized as free range farm and scale farm in Nanjing, Jiangsu, China, in different seasons. Additionally, a total of 350 sera of chickens were collected from free range farms to determine the presence of antibodies against T. gondii using sGRA8-ELISA.ResultsThe serological results showed that, antibodies were found in 194 of 250 (67.14%) samples from farms with T. gondii positive in soil and 41 of 100 samples from farms with T. gondii negative in soil (41.00%) (P < 0.01). The PCR detection of soil samples showed that, 7 (2.0%) of 350 samples collected from feeding zone in free range farms were found positive of T. gondii, whereas no sample was positive in scale farms. In the seasonal detections, T. gondii was found in 6 (3.33%) samples collected in autumn and 1 (0.56%) collected in winter.ConclusionsThe results indicated that the contamination of T. gondii in soil in the free range farms was higher than that in the scale farms and seroprevalence of T. gondii in chickens in the farm with soil contamination was higher than that with no soil contamination. The soil contamination might be an effective indicator of T. gondii infection in chickens.

Effects of Recombinant Toxoplasma gondii Citrate Synthase I on the Cellular Functions of Murine Macrophages In vitro

Toxoplasmosis, which is one of the most widespread zoonoses worldwide, has a high incidence and infection can result in severe disease in humans and livestock. Citrate synthase (CS) is a component of nearly all living cells that plays a vital role in the citric acid cycle, which is the central metabolic pathway of aerobic organisms. In the present study, the citrate synthase I gene of Toxoplasma gondii (T. gondii) (TgCSI) was cloned and characterized. The TgCSI gene had an open reading frame of 1665 bp nucleotides encoding a 555 amino acid protein with a molecular weight of 60 kDa. Using western blotting assay, the recombinant protein was successfully recognized by the sera of rats experimentally infected with T. gondii, while the native protein in the T. gondii tachyzoites was detected in sera from rats immunized with the recombinant protein of TgCSI. Binding of the protein to murine macrophages was confirmed by immuno fluorescence assay. Following incubation of macrophages with rTgCSI, the rTgCSI protein was found to have a dual function, with low concentrations (5–10 μg/mL) enhancing phagocytosis and high levels (80 μg/mL) inhibiting phagocytosis. Investigation of murine macrophage apoptosis illustrated that 5 μg/mL rTgCSI protein can significantly induce early apoptosis and late stage apoptosis (*p < 0.05), while 10 μg/mL rTgCSI protein significantly induced early apoptosis, but had no effect on late stage of apoptosis (**p < 0.01), and 80 μg/mL rTgCSI protein inhibited late stage apoptosis of macrophages (*p < 0.05). Cytokine detection revealed that the secretion of interleukin-10, interleukin-1β, transforming growth factor-β1 and tumor necrosis factor-α of macrophages increased after the cells were incubated with all concentration of rTgCSI, with the exception that 5 μg/mL rTgCSI had no effect on the secretion of interleukin-10 and interleukin-1β. However, secretion of NO and cell proliferation of the macrophages were substantially reduced. Taken together, these results suggested that TgCSI can affect the immune functions of murine macrophages by binding to the cells in vitro.

Investigation on the co-infections of Toxoplasma gondii with PRRSV, CSFV or PCV-2 in swine in part of China

Abstract The objective of the present investigation was to estimate the prevalence of Toxoplasma gondii infection and co-infection with porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV-2) in pigs in China. A total of 372 tissues or serum samples collected from pigs distributed in 9 provinces/municipalities of China during the period from February 2011 to November 2012 were assayed for T. gondii antigens and antibodies using enzyme linked immunosorbent assay (ELISA) technique, while the PCR was designed for the detection of the PRRSV, CSFV and PCV-2, respectively. The total positive rate of T. gondii, PRSSV, CSFV and PCV-2 was 9.14% (34/372), 50.00% (186/372), 37.10% (138/372) and 3.23% (12/372), respectively. Among the 34 T. gondii positive samples, 26 samples were simultaneously infected with T. gondii and viruses, while the remaining eight samples were infected with T. gondii alone. In addition, the co-infection rate of T. gondii with PRSSV, T. gondii with PRSSV and CSFV, T. gondii with PRSSV and PCV-2, T. gondii with CSFV and PCV-2, T. gondii with PRSSV, CSFV and PCV-2 was 1.61% (6/372), 4.03% (15/372), 0.27% (1/372), 0.27% (1/372) and 0.81% (3/372), respectively. The results of the present survey revealed that PRRSV and CSFV were the common pathogens co-existing with porcine toxoplasmosis in China, and both of them could increase the chances of T. gondii infection in pig. This is the first report of T. gondii co-infections with viruses in pigs. It is very important to understand the interactions of parasite and virus, and can be used as reference data for the control and prevention of co-infections of T. gondii and viruses in pigs.

Toxoplasma gondii Histone 4 Affects Some Functions of Murine Ana-1 Macrophages In Vitro

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan that can infect almost all nucleated cells. Histone proteins and DNA form the nucleosomes, which are the fundamental building blocks of eukaryotic chromatin. Histone 4 is an essential component of a histone octamer. In the present study, T. gondii histone 4 (TgH4) was cloned and the regulatory effect of TgH4 on murine macrophages was characterized. Bioinformatics analysis revealed that TgH4 was highly conserved in structure. Recombinant TgH4 (rTgH4) protein was identified by sera from rats experimentally infected with T. gondii and native TgH4 in the total soluble protein of T. gondii tachyzoites was recognized by polyclonal antibodies against rTgH4, as indicated by immunoblotting analysis. Immunofluorescence assay showed that TgH4 binds to macrophages. Following incubation with rTgH4, the toll‐like receptor 4 (TLR4) level of the macrophages was downregulated. Meanwhile, chemotaxis and the proliferation of macrophages were inhibited. However, rTgH4 can promote phagocytosis, apoptosis, and the secretion of nitric oxide, interleukin‐6, and tumor necrosis factor‐α from macrophages. Just 80 μg/ml rTgH4 can significantly elevate the secretion of interleukin‐10 and interleukin‐1β (p < 0.05 and p < 0.01). Viewed together, these outcomes indicated that rTgH4 can affect the functions of murine macrophages in vitro.

The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells

Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host–parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from Haemonchus contortus designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence assay, the binding activity of rHcSTP-1 was confirmed on surface of goat PBMCs, which resulted in expression of multiple cytokines and various immunoregulatory activities in vitro. The RT-PCR results showed that mRNA level of interleukin-2, TGF-β1, IFN-γ, and IL-17 (with 10 µg/ml) was upregulated and IL-10 was decreased. However, IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein. Further functional analysis showed that migratory activity of cells, intracellular nitrite production (NO), and apoptotic efficiency of PBMCs were elevated at significant level, whereas the proliferation of goat PBMCs and monocytes-associated major histocompatibility complex (MHC)-I and MHC-II expressions were decreased significantly at concentration-dependent fashion. Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells, which might represent a potential molecular target for controlling H. contortus infection in future.

The N- and C-terminal carbohydrate recognition domains of Haemonchus contortus galectin bind to distinct receptors of goat PBMC and contribute differently to its immunomodulatory functions in host-parasite interactions

BackgroundHco-gal-m is a tandem-repeat galectin isolated from the adult worm of Haemonchus contortus. A growing body of studies have demonstrated that Hco-gal-m could exert its immunomodulatory effects on host peripheral blood mononuclear cells (PBMC) to facilitate the immune evasion. Our previous work revealed that C-terminal and N-terminal carbohydrate recognition domains (CRD) of Hco-gal-m had different sugar binding abilities. However, whether different domains of Hco-gal-m account differently for its multiple immunomodulatory functions in the host-parasite interaction remains to be elucidated.ResultsWe found that the N-terminal CRD of Hco-gal-m (MNh) and the C-terminal CRD (MCh) could bind to goat peripheral blood mononuclear cells by distinct receptors: transmembrane protein 63A (TMEM63A) was a binding receptor of MNh, while transmembrane protein 147 (TMEM147) was a binding receptor of MCh. In addition, MCh was much more potent than MNh in inhibiting cell proliferation and inducing apoptosis, while MNh was much more effective in inhibiting NO production. Moreover, MNh could suppress the transcription of interferon-γ (IFN-γ), but MCh not.ConclusionsOur data suggested that these two CRDs of Hco-gal-m bind to distinct receptors and contributed differently to its ability to downregulate host immune response. These results will improve our understanding of galectins from parasitic nematodes contributing to the mechanism of parasitic immune evasion and continue to illustrate the diverse range of biological activities attributable to the galectin family.

Characterization of a secreted cystatin of the parasitic nematode Haemonchus contortus and its immune-modulatory effect on goat monocytes

BackgroundHaemonchosis is a disease of the small ruminant caused by a nematode parasite Haemonchus contortus, and it is most important and alarming challenges to the small ruminant’s production. The infection of the H. contortus could cause high economic losses worldwide. H. contortus is a blood feeding parasite which penetrates into the abomasal mucosa to feed the blood of the host and causing the anemia and decreased total plasma protein. Modulation and suppression of the immune response of the host by nematode parasites have been reported extensively, and the cysteine protease inhibitor (cystatin) is identified as one of the major immunomodulators.MethodsThe recombinant protein of HCcyst-3 was expressed in a histidine-tagged fusion soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, as well as papain, were identified by fluorogenic substrate analysis. Native HCcyst-3 protein was localized by an Immunohistochemical test. The immunomodulatory effects of HCcyst-3 on cytokine secretion, MHC molecule expression, NO production and phagocytosis were observed by co-incubation of rHCcyst-3 with goat monocytes.ResultsWe cloned and produced recombinant cystatin protein from H. contortus (rHCcyst-3) and investigated its immunomodulatory effects on goat monocyte. The rHCcyst-3 protein is biologically functional as shown by its ability to inhibit the protease activity of cathepsin L, cathepsin B, and papain. The immunohistochemical test demonstrated that the native HCcyst-3 protein was predominantly localized at the body surface and internal surface of the worm’s gut. We demonstrated that rHCcyst-3 could be distinguished by antisera from goat experimentally infected with H. contortus and could uptake by goat monocytes. The results showed that the engagement of rHCcyst-3 decreased the production of TNF-α, IL-1β and IL-12p40. However, it significantly increased the secretion of IL-10 and TGF-β1 in goat monocytes. After rHCcyst-3 exposure, the expression of MHC-II on goat monocytes was restricted. Moreover, rHCcyst-3 could upregulate LPS induced NO production of goat monocytes. Phagocytotic assay by FITC-dextran internalization showed that rHCcyst-3 inhibited the phagocytosis of goat monocytes.ConclusionsOur results suggested that the recombinant cystatin from H. contortus (rHCcyst-3) significantly modulated goat monocyte function in multiple aspects.