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.

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.

[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.

[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.

[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.

[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.

Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems.

In this study, we evaluated four methods to separate and purify Toxoplasma gondii tachyzoites from in vivo and in vitro culture systems, including trypsin digestion, purification with a 3-µm filter, CF-11 cellulose purification, and Percoll purification. Our results indicate that both purification with a 3-µm filter and CF11 cellulose purification methods remove leukocytes or HeLa cells, and can therefore be used as candidate methods for the purification of in vivo and in vitro culture products. Trypsin digestion had a high tachyzoite recovery rate, but 22.35% of leukocytes and 69.64% of HeLa cells remained in the purified products. Percoll solution [30% (v/v)] also had a high tachyzoite recovery rate, but 3.44% of leukocytes and 61.61% of HeLa cells remained in the purified products. The 40% Percoll solution was also a candidate method for purifying tachyzoites from in vivo culture products, with a 65.45% tachyzoite recovery rate and without leukocytes.

[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.

Efficacy of ginkgolic acids against Cryptosporidium andersoni in cell culture.

Cryptosporidium is a worldwide waterborne parasite and the treatment is a severe problem in immunocompromised patients. In this study, we used the in vitro culture system to evaluate the anti-Cryptosporidium activity of ginkgolic acids (GAs), nitazoxanide (NTZ), garlicin (GAR), and artemether (ART). The growth of Cryptosporidium andersoni in HCT-8 cells was determined by real-time PCR assay. When exposed to 5.00 µg/ml GAs or 10.00 µg/ml NTZ for 48 h, the number of C. andersoni in cultures was on a very low lever, but the number of parasites did not significantly decrease when exposed to GAR and ART. Our results indicate that GAs could be a potential drug for the treatment of cryptosporidiosis.

[Cloning, expression and immunodiagnostic evaluation of antigen EPC1 from Echinococcus granulosus].

OBJECTIVE: To clone and express EPCl gene of Echinococcus granulosus, and investigate its immunogenicity and diagnostic value. METHODS: Total RNA was extracted from hydatid cyst protoscoleces and EPC1 gene of Echinococcus granulosus was amplified by RT-PCR. The PCR product was cloned into pGEM-T vector, and then subcloned into the prokaryotic expression vector PET28a(+). The positive recombinants were transformed into Escherichia coli BL21 (DE3), and followed by expression of the protein induced by IPTG. The recombinant protein was identified by SDS-PAGE and Western blotting, and used to establish ELISA. Serum samples from patients with cystic echinococcosis (60 cases), alve-olar echinococcosis (37 cases), cysticercosis (16 cases), clonorchiasis sinensis (7 cases), schistosomiasis japonica (4 cases) and healthy persons (33 cases) were examined. RESULTS: The recombinant plasmid PET28a-EgEPC1 was identified by restriction enzyme digestion and sequencing. SDS-PAGE result showed that the recombinant containing recombinant plasmid PET28a-EgEPC1 expressed a soluble fission protein of EgEPC1 (about M, 11 000). The protein was recognized by pool sera of cystic echinococcosis patients. The overall sensitivity and specificity of diagnosis by ELISA for cystic echinococcosis were 78.3% (47/60), and 98.3% (59/60), respectively. The cross reaction with sera of alveolar echinococcosis was 40.5% (15/37). CONCLUSION: The recombinant EgEPC1 antigen has diagnostic value in cystic echinococcosis.

[Preliminary study on the immunological characteristics of permissive and non-permissive hosts infected with Schistosoma japonicum].

OBJECTIVE: To study the difference among immune responses of three kinds of experimental animals with different susceptibility to the infection of Schistosoma japonicum, and preliminarily explore the mechanism of the immune response in permissive and non-permissive hosts. METHODS: Twelve animals of each kind of rodents, C57BL/6 mice, Sprague Dawley (SD) rats and Microtus fortis, were randomly divided into the infected group and uninfected group each with 6 animals. In infected groups of C57BL/6 mice, SD rats, and M. fortis, each animal was infected with 20, 200 and 1000 cercariae of S. japonicum, respectively. 42 d later, all rodents were sacrificed. Adult worms in portal vein and granulomas in liver were observed and the sera were collected. The levels of cytokines IL-10 and IFN-gamma as well as serum IgG, IgG2a, and IgG1 were detected by ELISA. RESULTS: At the 42th day post infection, worms in portal vein and liver granulomas were observed in C57BL/6 mice and SD rats, but not in M. fortis. The level of IL-10 in the sera of SD rats [(2.21 +/- 0.12) pg/ml] was significantly higher than that in the sera of M. fortis [(1.64 +/- 0.39) pg/ml] and C57BL/6 mice [(0.10 +/- 0.04) pg/ml] (P<0.01). IL-10 in the sera of M. fortis was also significantly higher than that in the sera of C57BL/6 mice (P<0.01). IFN-gamma in the sera of SD rats [(0.21 +/- 0.11) pg/ml] was significantly higher than that in the sera of M. fortis [(0.11 +/- 0.03) pg/ml] and C57BL/6 mice [(0.09 +/- 0.02) pg/ml] (P<0.05), but no difference between M. fortis and C57BL/6 mice (P>0.05). The levels of IgG (1.53 +/- 0.31), IgG1 (1.48 +/- 0.44) and IgG2a (0.41 +/- 0.11) in SD rats were significantly higher than that in the sera of M. fortis (0.48 +/- 0.14, 0.15 +/- 0.03 and 0.12 +/- 0.061) (P<0.01). The levels of IgG (1.21 +/- 0.16), IgG1 (0.88 +/- 0.31) in C57BL/6 mice were significantly higher than that in the sera of M. fortis (P<0.01). IgG1 antibody is the predominant subclass in the three kinds of rodents. The levels of IL-10, IFN-gamma and antibody subclass IgG, IgG1, IgG2a in all non-infected rodents were not detected. CONCLUSION: IL-10 in non-permissive hosts, which is an essential agent in the regulation of Th2 immune response, is higher than that in permissive host It may play an important role in the resistance to schistosome in the non-permissive hosts.

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.

[Progress on the application of Cryptosporidium infected animal models and in vitro cultivation].

The genus Cryptosporidium is composed of protozoan parasites that infect epithelial cells in the microvillus border of the gastrointestinal tract of all classes of vertebrates, and cause severe diarrheal disease in a variety of neonatal animals, children and immunocompromised persons. Establishment of Cryptosporidium infected animal models and its in vitro cultivation system have established a good foundation for characterizing life cycle stage, exploring immunological mechanism, developing vaccines, screening and evaluating potential drugs, as well as assessing oocyst inactivation techniques. This paper reviews recent development and application of the Cryptosporidium infected animal models and its in vitro cultivation.

Effect of select medium supplements on in vitro development of Cryptosporidium andersoni in HCT-8 cells.

We evaluated the effect of fetal calf serum (FCS), glucose, ascorbic acid, calcium pantothenate, folic acid, and insulin on the growth of Cryptosporidium andersoni in human colon tumor (HCT-8) cells. After being incubated for 48 h, the proliferation of parasites was determined by real-time polymerase chain reaction (PCR) assay, and the development of C. andersoni was observed by transmission electron microscopy (TEM). Ten percent FCS was the best concentration for C. andersoni culture. Glucose, ascorbic acid, and insulin had a significant effect on the growth of C. andersoni when added into 10% FCS RPMI 1640. Calcium pantothenate had no significant effect and folic acid had the inhibited effect. We also observed the stages of trophozoite, macrogamont, microgamont, type I meront, type II meront, and sporozoite of C. andersoni in HCT-8 cells by TEM. Our results indicated that the best medium for C. andersoni was 10% FCS RPMI 1640 medium containing 50 mM glucose, 50 microg/ml ascorbic acid, and 0.3 U/ml insulin. Real-time PCR could provide a quick and precise technique to determine the proliferation of parasites. Cultivation of C. andersoni in HCT-8 cells will facilitate the study of interactions between parasites and host cells as well as provide a reliable system for evaluating anticryptosporidial compound efficacy.

[Identification of calf-origin Cryptosporidium bovis Shanghai isolate by nested PCR].

OBJECTIVE: To identify a strain of Cryptosporidium in the feces of naturally infected calf in Shanghai. METHODS: Stool sample was examined by modified acid-fast staining. The size and morphology of the oocysts were microscopically determined. Genomic DNA was extracted from the oocysts isolated from feces of a naturally Cryptosporidium-infected calf. According to the sequence of Cryptosporidium 18S rRNA gene, two pairs of primers were designed and synthesized. The PCR products was amplified by nested PCR and sequenced in double directions. Homology searches were done over the Web using the program Blast. Phylogenetic tree was constructed with NJ method by MEGA4.0 software. RESULTS: Oocysts of the Shanghai isolate were round or elliptical with a size of (5.6 +/- 0.49) microm x (5.2 +/- 0.51) microm. Nested PCR resulted in fragments of approximately 810 bp, and the 18S rRNA nucleotide sequence had 100% identity with C. bovis from Brazil (GenBank Accession No: 151935628). This isolate was clustered in the same clade with C. bovis from Brazil. It showed an identity of 99% with the sequences of C. bovis from Qinghai Province of China, Mongolia, USA, and Tunisia. CONCLUSION: The calf-origin Cryptosporidium derived from Shanghai has been identified as C. bovis.