Quantitative Microbial Risk Assessment of Cryptosporidium and Giardia in Public Drinking Water in China.

We aimed to assess the risks of Cryptosporidium and Giardia infections associated with drinking water for local residents, based on a quantitative microbial risk assessment, in three densely populated regions of China. In total, 45 source water samples and 45 treated water samples were collected from June to December 2014. Five Cryptosporidium-positive samples and 5 Giardia-positive samples were found. The annual probability of infection for individuals in Jintan (6.27 × 10 -4-2.05 × 10 -3 for Cryptosporidium and 7.18 × 10 -4-2.32 × 10 -3 for Giardia), Ezhou (6.27 × 10 -4-1.10 × 10 -2 for Cryptosporidium and 3.65 × 10 -4-1.20 × 10 -3 for Giardia), and Binyang (3.79 × 10 -4-1.25 × 10 -3 for Cryptosporidium) exceeded the tolerable risk of infection of 10 -4 set by the United States Environmental Protection Agency. Moreover, the corresponding disease burdens of cryptosporidiosis and giardiasis, due to direct drinking and residual water in these regions, exceeded the threshold of 10 -6 disability-adjusted life years per person per year set by the World Health Organization. These results provide insights into strategies to improve the safety of drinking water.

Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model.

BACKGROUND: Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. METHODS: The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. RESULTS: In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 µg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. CONCLUSIONS: Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

PCR-Based Detection of Cryptosporidium spp. and Enterocytozoon bieneusi in Farm-Raised and Free-Ranging Geese (Anser anser f. domestica) From Hainan Province of China: Natural Infection Rate and the Species or Genotype Distribution.

Cryptosporidium spp. and Enterocytozoon bieneusi are two important zoonotic pathogens that can infect humans and a broad range of animal hosts. However, few studies have been conducted to study infection of the two pathogens in domestic geese until now. The aims of the present study were to determine the prevalence of natural infection, and the species or genotype distribution of Cryptosporidium and E. bieneusi in farm-raised and free-ranging geese from Hainan Province of China. In total, 266 fecal samples of geese were collected (142 farm-raised and 124 free-ranging geese). Cryptosporidium spp. and E. bieneusi were identified by nested PCR and sequencing analysis of the SSU rRNA and the ITS region of the rRNA genes. A total of 4.1% (12/226) of the geese were positive for Cryptosporidium spp., with 0.7% identified in the farm-raised geese and 7.0% in the free-ranging geese. Two bird-adapted species/genotypes were identified: C. baileyi (n = 1) and Cryptosporidium goose genotype I (n = 11). Meanwhile, E. bieneusi was found in 13.9% (37/266) of geese, with 8.9% identified in the farm-raised and 21.8% in the free-ranging geese. Eleven genotypes of E. bieneusi were identified constituted with six known genotypes: D (n = 13), I (n = 5), CHG2 (n = 1), CHG3 (n = 5), and CHG5 (n = 1), and five novel genotypes named HNE-I to V (one each). All of the genotypes identified in the geese here belonged to zoonotic Groups 1 or 2. This study is the first to demonstrate the presence of Cryptosporidium spp. and E. bieneusi in domestic geese from Hainan, China, and provides baseline data that will be useful for controlling and preventing these pathogens in goose farms. The geese infected with E. bieneusi, but not with Cryptosporidium, should be considered potential public health threats.

Pro-Angiogenic Activity of Monocytic-Type Myeloid-Derived Suppressor Cells from Balb/C Mice Infected with Echinococcus Granulosus and the Regulatory Role of miRNAs.

BACKGROUND/AIMS: This study aims to predict the pro-angiogenic functions of monocytic-type myeloid-derived suppressor cells (M-MDSCs) derived from mice infected with Echinococcus granulosus. METHODS: M-MDSCs were collected from Balb/c mice infected with E. granulosus and normal mice (control) and cultured in vitro. Human umbilical vein endothelial cells (HUVECs) were stimulated with the cell supernatant, and angiogenesis was investigated and analysed by the Angiogenesis module of the software NIH Image J. RNA was extracted from fresh isolated M-MDSCs and analysed with miRNA microarray; differentially expressed miRNAs and their potential functions were analysed through several bioinformatics tools. Finally, quantitative PCR was used to confirm the results of microarray analysis. RESULTS: M-MDSCs from mice infected with E. granulosus could promote the formation of tubes from HUVECs in vitro. Moreover, vascular endothelial growth factor (VEGF) showed significantly high expression, whereas soluble fms-like tyrosine kinase-1 (sFlt-1) showed low expression at the transcriptional level in M-MDSCs from mice infected with E. granulosus. Microarray analysis of miRNAs showed that 28 miRNAs were differentially expressed in M-MDSCs from the two experimental mice groups, and 272 target genes were predicted using the microRNA databases TargetScan, PITA and microRNAorg. These target genes were mainly involved in the biological processes of intracellular protein transport, protein targeting to the lysosome and protein transport, and mainly located in the cytoplasm, neuronal cell body and membrane. Moreover, they were mainly involved in the molecular functions of protein binding, metal ion binding and SH3 domain binding. Further, the differentially expressed miRNAs were mainly enriched in the endocytosis, Wnt and axon guidance pathways, as well as the MAPK, focal adhesion, PI3K-Akt, cAMP, mTOR and TGF-ß signalling pathways, which are linked to immunoregulation and angiogenesis based on the results of bioinformatics analysis with DIANA-miRPath 3.0. In addition, the expression of eight miRNAs was randomly verified by quantitative PCR independently in three mice infected with E. granulosus and three normal mice. CONCLUSION: M-MDSCs have a potential angiogenic role during E. granulosus infection, and miRNAs may play a role in the immune response and angiogenesis functions of M-MDSCs through regulation of the identified signalling pathways.

The excretory-secretory products of Echinococcus granulosus protoscoleces stimulated IL-10 production in B cells via TLR-2 signaling.

BACKGROUND: Excretory-secretory products released by Echinococcus granulosus protoscoleces (EgPSC-ESPs) are well-known to regulate T cell responses. However, their direct influence on the differentiation of B cell subsets remains largely elusive. This study investigated the effects of EgPSC-ESPs on the differentiation of IL-10-producing B cells (B10), and explored the possible role of Toll-like receptor 2 (TLR-2) signaling in this process. RESULTS: In comparison to phosphate buffered saline (PBS), B cells exposed to the excretory-secretory products (ESPs) generated higher percentages of B10 cells, with higher expression of IL-10 mRNA, and larger amount of IL-10 production, which were in a dose dependent way. The mRNA and protein expression of TLR-2 in the ESPs-stimulated B cells were significantly higher than those in PBS, which was consistent to the results in B cells isolated from EgPSC infected mice. Moreover, TLR-2-/- B cells in response to ESPs stimulation expressed lower levels of IL-10 mRNA and produced undetectable IL-10 in comparison to those in normal B cells. In addition, Phosphatase and tensin homolog deleted on chromosome ten/AKT/Phosphatidylinositol-3 kinase (PTEN/AKT/PI3K) pathway was activated in ESPs-treated B cells, which was also dependent on TLR-2 signaling. Pam3CSK4, the agonist of TLR-2, could mock the effects of ESPs on the expression of PTEN, AKT and PI3K. CONCLUSION: Overall, this study revealed that TLR-2 signaling was required for B10 induction mediated by EgPSC-ESPs, which might be an immunomodulatory target against the parasite infection.

The excretory-secretory products of Echinococcus granulosus protoscoleces directly regulate the differentiation of B10, B17 and Th17 cells.

BACKGROUND: Excretory-secretory products (ESPs) released by helminths are well-known to regulate T cell responses in the host. However, their direct influence in the differentiation of naïve T cells, and especially B cells, remains largely unknown. This study investigated the effects of Echinococcus granulosus protoscoleces ESPs (EgPSC-ESPs) on the differentiation of IL-10-producing B cells (B10), IL-17A-producing B cells (B17) and Th17 cells. METHODS: BALB/c mice injected with EgPSC were used to evaluate the in vivo profiles of B10, B17 and Th17 cells. In vitro purified CD19+ B and naïve CD4+ T cells were cultured in the presence of native, heat-inactivated or periodate-treated EgPSC-ESPs, and the differentiation of these cell subsets were compared. RESULTS: In contrast to the control group, infected mice showed higher frequencies of B10, B17 and Th17 cells, and higher levels of IL-10 and IL-17A in the sera. Interestingly, B17 cells were first identified to express CD19+CD1dhigh. In vitro, B cells cultured with native ESPs exhibited a higher percentage of B10 cells but lower percentage of B17 and Th17 cells compared to the PBS group. Moreover, the relative expression of IL-10 and IL-17A mRNA were consistent with the altered frequencies. However, ESPs subjected to heat-inactivation or periodate treatment exhibited an inverse effect on the induction of these cell subsets. CONCLUSIONS: Our findings indicate that ESPs released by EgPSC can directly regulate the differentiation of B10, B17 and Th17 cells, which appear to be heat-labile and carbohydrate-dependent.

[Expression and Activity of Arginase from Monocytic-type Myeloid-derived Suppressor Cells in Rats Infected with Echinococcus granulosus].

Objective: To investigate the alteration of expression and activity of arginase from monocytic-type myeloid-derived suppressor cells（M-MDSC） in BALB/c mice infected with Echinococcus granulosus. Methods: Twelve BALB/c female mice were randomly divided into control and infected groups. The mice were injected intraperitoneally with 2 000 live protoscoleces or an equivalent volume of normal saline. After 120 days, peripheral blood was collected through venae orbitaeta, and mice were sacrificed for pathological examination. The spleen was collected under aseptic conditions and single-cell suspension was prepared for M-MDSC isolation using the magnetic bead separation technology. Total RNA was extracted from M-MDSC, cDNA was generated, and genes with differential expression without and with infection were screened using the chip hybridization method. The resulting genes were further validated using real-time PCR. The activity of arginase from peripheral blood was also measured. Results: Single cyst was formed within the abdomen and internal organs 120 days after infection. Chip hybridization and real-time PCR showed that the relative expression of arginase from M-MDSC in the infected group （7.92±0.85 and 11.97±5.39, respectively） was significantly higher than that in the control group （1.65±0.19 and 1.00±0.57, respectively） （P<0.05）. The activity of arginase was also significantly higher in the infected group ［（3.83±0.44）U/Lï¼½ than in the control [(1.57±0.57）U/L]. Conclusion: The expression and activity of arginase from mouse M-MDSC both increase significantly after infection with Echinococcus granulosus.

[T Follicular Helper Cells and Related Molecules in Schistosoma japonicum Infected Mice after Praziquantel Treatment].

Objective: To investigate the pathological changes of liver and spleen of mice infected with Schistosoma japonicum and the changes of T follicular helper (Tfh) cells and surface molecules after praziquantel treatment. Methods: Fifteen female C57BL/6 mice （6-8 weeks） were randomly assigned into the praziquantel treated infection group （treated group）, infection control group (untreated group) and uninfected group （n=5 in each group）. The mice in the treated group and untreated group were each infected with 20 S. japonicum cercariae through the abdominal skin, and mice in the treated group were further administered with intragastric praziquantel [200 mg/（kg·d）] at week 6 post-infection for 3 consecutive days. Mice were sacrificed at week 4 after treatment to observe the morphological changes of liver and spleen and calculate the worm reduction rate and the liver egg reduction rate. The Tfh cell to CD4+ T cell ratio, as well as the expression of inducible T-cell costimulator （ICOS） and programmed cell death protein 1（PD-1） on peripheral blood and spleen, were determined by flow cytometry. Schistosome soluble egg antigen (SEA) specific IgG antibodies in serum were detected by ELISA. Results: The pathological changes of liver and spleen in the treated group were less severe compared with those of the untreated group, with a worm reduction rate of 84.1% and liver egg reduction rate of 69.1%. Flow cytometry showed that the percent of Tfh cells in peripheral blood and spleen was significantly higher in the treated group（14.7%-18.0%, 15.6%-25.0%） and the untreated group（13.7%-16.7%, 12.4%-18.2%） than that in the uninfected group（2.5%-6.8%, 4.9%-8.0%）, but there was no significant difference between the treated and untreated groups. The expression of ICOS in the peripheral blood and the spleen was significantly higher in untreated group（1.3%-3.2%, 4.1%-7.0%） than in the treated group（0.7%-1.1%, 1.8%-6.8%） and the uninfected group（0.2%-0.3%, 0.5%-0.8%）（P<0.01）, The expression of ICOS in the spleen was significantly higher in the treated group than in the uninfected group (P<0.01), while this difference was not found for ICOS expression in the peripheral blood. The PD-1 expression in the peripheral blood and spleen was significantly higher in the untreated group（0.8%-1.9%, 4.1%-10.7%） than in the uninfected group（0.4%-0.8%, 1.2%-1.8%）（P<0.01）, while there was no significant difference between the treated group（0.5%-1.5%, 4.5%-8.9%） and the untreated group (P>0.05). In addition, there was no significant difference in the level of SEA specific IgG between the treated group（2.015±0.061） and the untreated group（1.969±0.038） at 4 weeks after praziquantel treatment. Conclusion: Praziquantel treatment can significantly alleviate the lesions of the liver and the spleen and decrease the ICOS expression by Tfh cells in the peripheral blood and spleen.

[Serological Analysis of Antibodies against Cysticercus cellulosae and Toxoplasma gondii in 122 Patients with Meningitis Encephalitis Syndrome].

Objective: To examine the IgG and IgM antibodies for parasites Cysticercus cellulosae and Toxoplasma gondii in 122 patients with meningitis encephalitis syndrome, and provide basis for clinical diagnosis of the meningitis encephalitis syndrome. Methods: The sera were collected from patients with meningitis encephalitis syndrome in Shanghai Jiaotong University Affiliated Sixth People's Hospital, People's Hospital of Danyang City, and Jiangsu University Affiliated Hospital from August, 2014 to December, 2015. Serum IgG and IgM antibodies for cysticercus and T. gondii were examined using antibody test kits. The antibody positive rate was calculated and its distribution was analyzed by gender, season, age and occupation. Results: A total of 122 patients with meningitis encephalitis syndrome were included. Seventeen and 22 patients of them were positive for IgG （13.9%, 17/122） and IgM（18.0%, 22/122） against cysticercus, respectively, while 29 and 8 cases were positive for IgG （23.8%, 29/122） and IgM （6.6%, 8/122） against T. gondii. The positive rate of cysticercus and T. gondii in males was 30.6%（22/72） and 31.9%（23/72） respectively, while that in females was 26.0%（13/50） and 24.0% （12/50）. The positive rate of IgM against cysticercus was 12.0%（3/25）, 27.0%（17/63）, 6.9% （2/29）, and 0（0/5） from spring to winter, highest within 13-25 years（45.0%, 9/20） among age groups, and highest in workers（7/14） among various occupations. The positive rate of IgM against T. gondii was 4.0%（1/25）, 11.1% （7/63）, 0（0/29）, and 0（0/5） from spring to winter, highest in ages >65 years（44.0%, 11/25）, and highest in patients with other occupations（4/10）. There was no statistically significant difference in the positive rate between males and females, and among different seasons, ages and occupations. Conclusion: The positive rate of antibodies against cysticercus and T. gondii is high in the patients included, suggesting that a serological test for parasite infection might be performed during clinical diagnosis of meningitis encephalitis syndrome.

[Changes of Phenotype and Phagocytosis of Peritoneal Macrophages in Mice Infected with the Larval-stage of Echinococcus granulosus].

Objective: To investigate the phenotype and phagocytosis changes of the peritoneal macrophages (Mφ) in mice infected with the larval-stage Echinococcus granulosus, and explore the role of Mφ in the responses to parasite infection. Methods: Twenty-four female BALB/c mice (age of 6-8 weeks) were randomly assigned into control group and infection group (n=12 in each group). The mice in the infection group were intraperitoneally injected with 2 000 protoscoleces, while the control mice were injected with equal volume of PBS. Five months after infection, the peritoneal mononuclear cells were collected, and the percentage of Mφ and the expression of surface markers CD40, CD80, CD86, and major histocompatibility complex Ⅱ (MHCⅡ) were determined by flow cytometry. The absorbance（A490 value） of Mφ at different concentrations（1×106, 5×105, 1×105） was determined by the neutral red assay to evaluate the phagocytic ability of Mφ. Results: The Mφ constituted（30.40±3.15）% and（20.75±5.91）% in mononuclear cells in the infection and the control groups, respectively. The percentages of Mφ expressing CD40, CD80, CD86, and MHC Ⅱ were（45.33±5.51）%, （61.00±10.61）%, （56.88±10.66）% and （27.00±3.82）% in the infection group, which were all significantly higher than those in the control ［（41.43±6.19）%, （59.23±8.65）%, （10.91±1.82）% and （13.67±3.01%）］ （P<0.05）. The A490 values of Mφ at 1×106, 5×105, 1×105 were 0.41±0.03， 0.24±0.05 and 0.16±0.01 in the infection group, which were significantly lower than those in the control （0.61±0.15, 0.47±0.07 and 0.18±0.01）（P<0.01）. Conclusion: The phagocytic ability of peritoneal Mφ is dramatically weakened after infection, but the expression of activation-associated surface markers is significantly up-regulated after infection.

[Molecular characteristics and RNA interference efficiency of Schistosoma japonicum Sj79 gene].

OBJECTIVE: To study the structural features and characteristics of a novel gene Schistosoma japonicum 79 (Sj79), and observe its effect of RNA interference (RNAi) , so as to provide the experimental basis for its further function study and mechanism study of anti reproductive development of schistosome. METHODS: The gene structure and characteristics of Sj79 were analyzed by bioinformatics methods. Then the expressions of Sj79 messenger RNA (mRNA) during the different developmental stages of schistosome were analyzed and the effects of RNAi silencing were observed by the soaking method. The transcriptional levels of Sj79 after RNAi were detected by real time PCR. RESULTS: The open reading frame of Sj79 contained 696 base pairs with an exon structure. The gene had obvious stage specificity, and its transcriptional level in mature female worms was the highest. After soaking for 3 d, the Sj79 mRNA level [ (41.0 ± 12.3)%] in the siRNA-1 group with low dosage (20 nmol/L) was lower than that in the siRNA-NC group [(103.2 ± 14.4)%], the difference was statistically significant (t = 3.28,P < 0.05). When with high dosage (200 nmol/L), both the Sj79 mRNA levels in the siRNA-1 group [(15.8 ± 10.9)%] and siRNA-2 group [(11.1 ± 8.8)%] were significantly lower than that in the siRNA-NC group [(100.1 ± 6.3)%] (t = 13.44, 27.84, both P < 0.01). After soaking for 7 d, only the Sj79 mRNA levels in the siRNA-1 group [(43.4 ± 4.5)%] and siRNA-2 group [(62.5 ± 5.4)%] with low dosage were lower than that in the siRNA-NC group [(100.4 ± 5.2)%], and the differences had statistical significance (t = 8.33, 5.07, both P < 0.01). CONCLUSION: Through this study, we have improved the mRNA sequence and genomic information of Sj79 gene, and understood its structural features, as well as selected out two effect fragments siRNA-1 and siRNA-2, which will provide the basic evidences for the further study on egg laying interference of the female adult worm of schistosome in vitro.

[Infection and molecular characteristics of Giardia in clinical diarrheal patients].

OBJECTIVE: To initially understand the infection status and the molecular characteristics of Giardia in clinical diarrheal patients. METHODS: A total of 95 stool samples were collected from the clinical diarrheal patients admitted in a hospital in Shanghai from May to July, 2014, and the Giardia cysts in the samples were examined by an optical microscope. Then the tpi gene of Giardia in the positive samples were amplified by using the nested-PCR method, and the PCR products were sequenced and analyzed by using BLAST, ClustalX 1.83, and the phylogenetic tree was drawn by using MEGA6.0 software. RESULTS: Only one patient was infected with Giardia and the positive detection rate was 1.05%. The Giardia cysts in the fecal specimen were seen clearly under the microscope. Through the identification by PCR, the amplified fragment was about 530 bp, and the sequencing analysis indicated it was Giardia and which was further identified as assemblage B by drawing phylogenetic tree based on tpi gene. Meanwhile, the sequence had 100% homology with the reported sequence from huian (KF271445). CONCLUSIONS: Giardia infection can occur in the clinical diarrheal patients. The study could provide more data for understanding the genetic characteristics of Giardia and the epidemiological study of giardiasis.

[Expression of Cocktail DNA Vaccine Comprising Toxoplasma gondii SAGI, MIC3 and ROP2 Using Fluorescent Protein-Reporting Vectors and Evaluation of Its Immunogenicity].

OBJECTIVE: To construct a cocktail DNA vaccine that expresses multiple genes of Toxoplasma gondii and investigate its immunogenicity in mice. METHODS: Genes for surface antigens (SAG), microneme(MIC), and rhoptry protein(ROP) were amplified from genomic DNA of T. gondii and then cloned separately into eukaryotic fluorescent protein expression vectors pShuttle-CMV-MCS-EFlα-AmCyan, pLVX-IRES-Zsgreen and pLVX-IRES-rfp, to construct expression plasmids pShuttle-SAG1, pLVX-Zsgreen-MIC3 and pLVX-rfp-ROP2. 293F cells were transfected with a combination of the three plasmids using the polyethylenimine (PEI) method. Forty-eight hours later, the expression of the three genes was observed under a fluorescence microscope. In addition, 30 C57BL/6 mice were randomized to receive intramuscular injection of saline (50 pA, group A), pShuttle+pLVX-Zsgreen+pLVX-rfp empty plasmids(2 µg/µL, 17 µL of each, group B) and pShuttle-SAG1+pLVX-Zsgreen-MIC3+ pLVX-rfp-ROP2 recombinant plasmid (2 µg/µL, 17 µL of each, group C). After 28 days, anti-T. gondii antibody in mouse serum was detected by ELISA, to evaluate the immunogenicity of the vaccine. RESULTS: The SAG1, MIC3 and ROP2 genes were amplified from the genomic DNA, with product sizes of 1, 1.1 and 1.7 kb. The eukaryotic expression plasmids pShuttle-SAG1, pLVX-Zsgreen-MIC3 and pLVX-rfp-ROP2 were constructed, and the corresponding fluorescences (blue, green and red) were observed after transfection. On day 28 after mouse vaccination, ELISA showed that the mean A(450) values for serum IgG in groups A, B and C were (0.620±0.029), (0.741±0.040) and (1.561±0.131), respectively, with the group C value being significantly higher than the others (P<0.01). CONCLUSION: The cocktail DNA vaccine comprising T. gondii SAG1, MIC3 and ROP2 shows promising immunogenicity in mice, and the fluorescent protein expression vectors are reliable tools for expression of the target genes.

[Effect of toll-like receptor (TLR) 7 deficiencies on the in vivo immune response against Schistosoma japonicum].

OBJECTIVE: To explore the toll-like receptor 7 knocked out (TLR7-/-) mice immune response against Schistosoma japonicum. METHODS: C57BL/6 mice (WT) and TLR7-/- mice (TLR7-/-) were infected with 20 S. japonicum cercariae via shaved abdomen. There were nine mice in each group. At 6 weeks post-infection, mice were sacrificed. Adult worms were harvested by perfusion of the portal venous system, and the number of adult worms was determined. At the time of perfusion, livers were collected, weighed, and digested overnight with 5% potassium hydroxide, and eggs were counted. In addition, spleens were aseptically harvested when WT and TLR7-/- mice were sacrificed at day zero and 6 weeks after S. japonicum infection. After 72 hours of the co-culture with or without S. japonicum eggs, the culture supernatants were collected for cytokine assays by ELISA assay. RESULTS: At 6 weeks after infection, there was no significant difference in number of worms [(10.5 +/- 3.3) vs (9.8 +/- 5.2)] and eggs per gram of liver tissue [(38 251.9 +/- 4 891.5) vs (38 160.9 +/- 3 341.0)] between WT and TLR7-/- mice. As for Th1/Th2 cytokine secretion from spleen cells, the levels of TNF-alpha [(43.7 +/- 9.8) pg/ml] and INF-gamma [(215.2 +/- 35.4) pg/ml] from TLR7-/- infected mice were lower than those of WT infected mice[(63.4 +/- 22.9) pg/ml, (383.5 +/- 253.3) pg/ml]. For Th2 cytokines detection, the production of IL-10 [(1702.6 +/- 572.3) pg/ml] and IL-4 [(59.5 +/- 10.1) pg/ml] from TLR7-/- mice were higher than those of WT mice [(595.2 +/- 386.3) pg/ml, (8.3 +/- 0.9) pg/ml] (P < 0.05, P < 0.01), while IL-4 level [(63.9 +/- 33.9) pg/ml] from TLR7-/- infected mice was higher than those of WT infected mice [(23.3 +/- 11.5) pg/ml]. CONCLUSION: TLR7-/- mice has a dominant Th2 response under the normal state. The absence of TLR7 does not influence the immune response against S. japonicum infection at 6 weeks post-infection.

[Accumulation of myeloid-derived suppressor cells in the spleen and peripheral blood of Schistosoma japonicum-infected mice].

OBJECTIVE: To determine the accumulation of CD11b+ Gr-1+ myeloid-derived suppressor cells (MDSC) in Schistosorna japonicum-infected mice. METHODS: Twenty-four C57BL/6 mice were infected cutaneously with S. japonicum cercariae. Peripheral blood samples were collected at 1, 2, 6 and 8 weeks post-infection (6 mice for each group). At 6 and 8 weeks post-infection, spleens were removed and a single-cell suspension was prepared. At the same time, 6 healthy mice each served as control. During the different stages of infection, the levels of MDSC, Gr-1+ cells, CD11b+ cells in murine peripheral blood and spleen were detected by flow cytometry. The possible function of MDSC on T cells was evaluated by using a CCK-8 method and CFSE proliferation assay. RESULTS: At 6 and 8 weeks post-infection, the levels of MDSC (38.2%-57.8% and 47.1-77.6%, respectively), Gr-1+ cells (28.9%-44.6%, 40.4%-72.9%), and CD11b+ cells (36.0%-48.1%, 40.3%-68.3%) in infection group were significantly higher than that of the controls (15.1%-20.4%, 8.4%-17.3%, 9.8%-22.6%), and that of infection group at 1 week (16.2%-19.8%, 13.0%-16.8%, 17.6%-19.4%) and 2 weeks (19.8%-29.5%, 17.2%-22.2%, 20.9%-33.3%) post-infection (P < 0.01). No significant difference was found in the levels of MDSC, Gr-1+ cells, CD11b+ cells among infection group at 1 and 2 weeks post-infection and control group. Moreover, the fluctuation trends of these cells in the spleens of infected mice were similar to those cells in peripheral blood (P > 0.05). Strikingly, the proliferation index of normal CD4 T cells was significantly lower after co-culture with Gr-1+ cells isolated from infected mice. CONCLUSION: Schistosoma japonicum infection induces higher level of MDSC in mice, and Gr-1+ cells isolated from the infected mice can significantly inhibit the proliferation of the normal CD4+ T cells.

Age-related infection with Cryptosporidium species and genotype in pigs in China.

OBJECTIVE: Pigs, as hosts of zoonotic Cryptosporidium species/genotypes, are domestic animals with public health significance. The present study was to characterize the infection rate and species/genotype of Cryptosporidium in pre-weaned and post-weaned pigs from Shanghai and Shaoxing, China. METHODS: A total of 208 fecal samples (42 from pre-weaned piglets, and 166 from post-weaned pigs) were examined by nested PCR of the 18S rRNA gene and analyzed by phylogenetic DNA fragment sequencing of secondary PCR products. RESULTS: Infection was detected in 79 samples (19/42 pre-weaned piglets, and 60/166 post-weaned pigs). C. suis (14/79) and Cryptosporidium pig genotype II (65/79) were identified; piglets were more susceptible to the former (13/14) and post-weaned pigs to the latter (59/65). CONCLUSION: Infection of Cryptosporidium spp. in pigs was age-specific; piglets were more susceptible to C. suis while pigs were more susceptible to Cryptosporidium pig genotype II. These findings combined with the isolation of the two Cryptosporidium from water suggest that pigs may be a source of zoonotic Cryptosporidium water pollution. Improvements in pig feeding practices, sewage discharge, feces disposal and field worker protection are therefore important to prevent potential public health problems.

Surveillance on the status of immune cells after Echinnococcus granulosus protoscoleces infection in Balb/c mice.

BACKGROUND: Cystic echinococcosis is a global parasitic disease caused by infection with Echinococcus granulosus larvae with potentially life-threatening complications in humans. To date, the status of the immune cells believed to be associated with the pathogenicity of E. granulosus infection has not been demonstrated clearly. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we developed a multiplex flow cytometry assay to investigate the systemic immune status of innate and adaptive immunity at 30, 180, 360 days post-infection (dpi) in mice infected with E. granulousus. At 30 dpi, an increase in the number of CD11b(+) and CD11c(+) antigen-presenting cells (APCs) was observed. This was accompanied by the slight down-regulated expression of the co-stimulatory molecule MHC-II, indicating the impairment of APCs in early infection through the release of secretory-excretory products. In all infected groups, we observed a significant increase in innate immune cells, including APCs and GR-1(+) cells, and a dramatic increase in the myeloid-derived suppressor cells (MDSC) expressing CD11b(+)/GR-1(+). Moreover, the upregulation of the activated markers CD69, CD44, CD40L, and the downregulation of CD62L were observed in the CD4(+) and CD8(+) T cells following infection. Regulatory T cells expressing CD4(+)/CD25(+)/FoxP3 (+) increased significantly over the course of infection. CONCLUSIONS: Our findings demonstrate that the microenvironment in the peripheral immune system after E. granulosus infection changes in subtle but detectably ways, especially during the persistent period of infection. We found that T cells were activated following infection, but observed that the significant increase of immunosuppressive cells such as MDSC and Treg cells could inhibit T cell response to E. granulosus antigens. We suggest these cells may play a neglected but key role in the downregulation of the immune response in long-term parasitic infection. Understanding the basic functions and temporal interactions of these immunosuppressive cells will pave the way for new strategies of parasite vaccine design.

[Research progress on the biology and epidemiology of Cyclospora cayetanensis].

Cyclosporiasis is one of the emerging parasitic diseases. Cyclospora cayetanensis is so far the only species infecting humans in the Cyclospora genus. This paper reviews mainly the biological characteristics of C. cayetanensis and the current epidemiology status of human infection.

[Cloning, expression and bioinformatics analysis of cyclophilin of Echinococcus granulosus].

OBJECTIVE: To clone and express EgCyP gene of Echinococcus granulosas and analyze EgCyP using bioinformatics. METHODS: Total RNAS of adult E. granulosus was extracted and reversedly transcripted to cDNA. EgCyP gene was amplified from cDNA and inserted into vector pET28a. Recombinant plasmid pET28a-EgCyP was transformed into E. coli BL21 (DE3) for expression under the induction of IPTG. The expressed product was identified by SDS-PAGE and Western blotting. EgCyP was analyzed by the bioinformatics software. RESULTS: The EgCyP gene was successfully amplified from cDNA of adult E. granulosus and a fusion protein was expressed in E .coli BL21 (DE3). The molecular weight of the expressed protein was about 22 kDa. The Western blotting indicated that the antigenicity of the protein was specific. The bioinformatics analysis revealed that there were 7 antigen epitopes in EgCyP. CONCLUSION: EgCyP of E. granulosus is cloned and expressed in E. coli BL21 (DE3) successfully, which might be the foundation for the further study of its immunogenicity.