Table of Content

30 June 2018, Volume 36 Issue 3

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The progress of national malaria elimination and epidemiological characteristics of malaria in China in 2017

ZHANG Li, FENG Jun, ZHANG Shao-sen, XIA Zhi-gui*, ZHOU Shui-sen

2018, 36(3):  2-201-209. 

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Objective To analyze the progress of malaria elimination and epidemiological characteristics of malaria in China in 2017, in order to provide evidence-based reference for implementation of malaria elimination program. Methods The malaria epidemic data in 31 provinces/municipalities/autonomous regions(Taiwan, Hongkong and Macao regions not included) were collected through the Malaria Annual Reporting System in 2017. The epidemic situation, population distribution, species classification, regional distribution and infection sources were analyzed in Microsoft Excel 2010 software.  Results There were accumulative 2 861 malaria cases reported by 828 institutions in 2017, decreased by 13.9% compared with 2016 (3 321 cases), comprising 2 675 Chinese cases (93.5%) and 186 foreigner cases (6.5%). The male-to-female ratio was 11.4 ∶ 1. The cases were concentrated at the age range of 20-49 years (80.8%, 2 311/2 861). Nine were clinically diagnosed patients (0.3%, 9/2 861). There were 537 Plasmodium vivax cases (20.2%, 537/2 861), 1 822 P. falciparum cases(63.7%, 1 822/2 861), 67 P. malariae cases (2.3%, 64/2 861), 352 P. ovale cases (12.3%, 352/2 861), 37 cases of mixed-infection (1.3%, 37/2 861) and one P. knowlesi case. None was indigenously infected, 2 858 (99.9%, 2 858/2 861) were imported from other countries, and 3 (0.1%, 3/2 861) were infected due to transfusion. The top 5 provinces with regard to the number of malaria cases were Guangxi (13.4%, 382/2 861), Yunnan (11.4%, 325/2 861), Jiangsu (8.4%, 239/2 861), Shandong (7.3%, 209/2 861) and Sichuan (7.3%, 209/2 861). Furthermore, 136 cases(4.8%, 136/2 861) with severe clinical symptoms were reported in 18 provinces/municipalities/autonomous regions, and 7 deaths (0.2%, 7/2 861) were reported in 6 provinces/municipalities/autonomous regions. Case reportings were done within 24 h for all the cases, 83.7% (2 396/2 861) had completed case investigations within three days.  Conclusion This is the first time that no indigenous cases were reported in China in 2017. Continued efforts are needed to strengthen the monitoring and management of imported cases from abroad in some key regions to prevent malaria retransmission.

Analysis of gene sequence polymorphisms and prediction of antigen epitopes of merozoite surface protein-3 in Plasmodium falciparum from different infection sources

DONG Ying1, DENG Yan1, XU Yan-chun1, CHEN Meng-ni1, MAO Xiang-hua1,

2018, 36(3):  3-210-217. 

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Objective To analyze the gene sequence polymorphisms and predict antigen epitope of Plasmodium falciparum merozoite surface protein-3 (PfMSP-3) from different infection sources of P. falciparum in Yunnan Province. Methods Blood samples on filter paper and the corresponding epidemiology data of patients with falciparum malaria reported through the Reporting System of Chinese Center for Disease Control and Prevention, were collected from August 2012 to December 2016. P. falciparum DNA was extracted, and the PfMSP-3 gene was amplified with semi-nest PCR. PCR products were sequenced, and all sequences were aligned with the reference sequences LN999944.1 of Pf3D7 isolate and U08851.1 of PfK1 isolate in GenBank. The haplotype number, expected heterozygsity (He), and genetic differentiation (Fst) of PfMSP-3 gene in different Plasmodium populations were analyzed by MEGA 5.04 and Arlequin 3.5.2.2 softwares. The haplotype media evolution tree was constructed with the Network 4.6.0 software. The mean number of non-synonymous substitutions per non-synonymous site (Ka) and synonymous mutations per synonymous site (Ks) were calculated with the DnaSP 5.10 software. T/B cell epitopes of PfMSP-3 peptides were analyzed and predicted using the IEDB online software.  Results A total of 190 blood samples were collected, of which 181 produced PfMSP-3 fragments at 1 100 bp, and 167 PCR products were sequenced successfully, including 121 from Myanmar, 37 from Africa and 9 from indigenous cases in Yunnan. Alignment results showed that there were 36 haplotypes in the 167 DNA sequences, with an He of (0.409 + 0.183) and a Ka/Ks of 0.98. The 36 haplotypes evolved along the direction from Pf3D7 type (Ⅰ group), to transitional type (Ⅱ group), then PfK1 (Ⅲ group). Among them, 7 haplotypes including H_1 and H_9 were Pf3D7 type, 6 haplotypes including H_7 and H_8 were the transitional type, and 23 haplotypes including H_2 and H_3 were the PfK1 type. The Pf3D7 type, transitional type and PfK1 type accounted for 49.7% (83/167), 12.6% (21/167) and 37.7% (63/167), respectively. The genetic differentiation of PfMSP-3 was highest between African and Myanmar P. falciparum populations (Fst = 0.049) and lowest between African and Yunnan populations (Fst = 0.032). The peptide chains of 36 haplotypes of PfMSP-3 had a high hydrophilicity than hydrophobicity, with indexes of 1.050~2.535 and -2.583~-3.544, respectively. The activity score of T cell epitope was -1.1. The average activity score of B cells was > 0.5, more specifically ranked as Pf3D7 type ＞ transitional type ＞ PfK1 type.  Conclusion The PfMSP-3 gene of P. falciparum populations from different infection sources has evolved into 3 genotypes, and the B cell epitopes are predicted to be more active than T cell epitopes in PfMSP-3 proteins of all genotypes.
  

Expression of the regulatory T cell transcription factor Foxp3 and interleukin-8 in liver tissues of patients with echinococcosis

SHAN Jiao-yu1,2, REBIYA·Nuli1, LI Rui1, LIN Ren-yong2, WEN Hao2,3, LI Hai-tao2,3*

2018, 36(3):  4-218-223. 

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Objective To explore the expression of the regulatory T cell transcription factor Foxp3 and interleukin-8(IL-8) in liver lesions in patients with echinococcosis. Methods Liver tissues were collected from 16 patients with alveolar echinococcosis(AE), 23 patients with cystic echinococcosis(CE) and 8 patients with hepatic hemangioma. HE staining and immunohistochemical(IHC) analysis were performed to examine Foxp3 in liver tissue. Western blotting was performed to detect protein levels of Foxp3. Total RNA was extracted from liver tissue by Trizol, and the mRNA levels of Foxp3 and IL-8 were tested by Real-time PCR. The correlation between Foxp3 and IL-8 was analzyed by Spearman method. Between-group comparisons were made by t test. 　Results  H&E staining results showed incomplete horny layer and growth layer of the liver in AE cases, a thick horny layer in CE cases, and complete liver structures in hepatic hemangioma cases. Liver IHC results showed abundant cells positive for Foxp3 in the AE group, in contrast with few in the CE group and absence in  the hepatic hemangioma cases. Western blotting analysis revealed that the gray values of Foxp3 were (0.977 ± 0.082), (0.728 ± 0.094), and (0.290 ± 0.084), respectively in the AE, CE and hepatic hemangioma groups. There were significant differences in the gray value between AE or CE group and the hepatic hemangioma group(both P < 0.05), but there was no difference between AE and CE groups（P > 0.05）. Results of real-time PCR showed that the relative levels of Foxp3 mRNA were(0.013 ± 0.003), (0.007 ± 0.002), and (0.004 ± 0.001), respectively in the AE, CE and hepatic hemangioma groups. There was a significant difference in the Foxp3 mRNA level between AE and hepatic hemangioma groups (P < 0.05), but no difference was found for CE versus AE or CE versus hepatic hemangioma(P > 0.05). The IL-8 mRNA levels were(0.402 ± 0.068), (0.005 ± 0.019), and(0.002 ± 0.001), respectively in the AE, CE and hepatic hemangioma groups, with significant differences for all the between-group comparisons(P < 0.01). The Spearman correlation analysis demonstrated that the Foxp3 and IL-8 mRNA levels were positively correlated in the AE group (r = 0.802, P < 0.01).  Conclusion Foxp3 and IL-8 mRNA in the liver tissue are both higher in AE than in CE, suggesting that AE may stimulate secretion of a large amount of IL-8, which, together with Foxp3, causes severe inflammation at the foci.

Changes of myeloid-derived suppressor cells and Th17 cells in mice infected with Echinococcus granulosus protoscoleces

YU Ai-ping, YIN Jian-hai, GONG Wen-ci, CAO Sheng-kui, CAO Jian-ping, SHEN Yu-juan*

2018, 36(3):  5-224-230. 

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Objective To investigate the changes of the myeloid-derived suppressor cells (MDSCs) and T helper 17 (Th17) cells in mice infected with Echinococcus granulosus protoscoleces and its implications. Methods Twenty female BALB/c mice of 6 weeks were randomly divided into the infection and normal control groups, each with 10 mice. The mice in the infected group were intraperitoneally injected with 2 000 protoscoleces, and those in the control group were injected with a same volume of normal saline. Spleen cells, peripheral blood leukocytes, and peritoneal cells were collected eight months after infection, in which the percentages of MDSCs, their subpopulations (M-MDSCs and PMN-MDSCs) and Th17 cells were assessed by flow cytometry. The correlations of MDSCs and their subpopulations with Th17 cells were determined by the Pearson correlation analysis.  Results In the infection group, the proportion of MDSCs in spleen cells, peripheral blood leukocytes, and peritoneal cells were（14.72 ± 4.27）%, （57.04 ± 6.78）% and（15.35 ± 5.56）%, respectively, all significantly higher than those in the control group ［(8.84 ± 2.12)%, (30.53 ± 1.58)% and (1.74 ± 0.63)%, respectively］ (P < 0.05). In the infection group, the proportion of M-MDSCs in spleen cells, peripheral blood leukocytes, and peritoneal cells were (1.29 ± 0.24)%, (6.27 ± 2.11)% and(2.14 ± 0.94)%, respectively, all significantly higher than those in the control group[(0.72 ± 0.25)%, (2.11 ± 1.27)%, (0.25 ± 0.06)%](P < 0.05). The proportion of PMN-MDSCs in spleen cells, peripheral blood leukocytes, and peritoneal cells were (9.31 ± 2.65)%, (46.72 ± 5.67)%, (7.06 ± 2.36)% in the infection group, and were (7.07 ± 3.20)%, (25.42 ± 2.05)%, (1.08 ± 0.40)% in the control group, the proportion of PMN-MDSCs in peripheral blood leukocytes and peritoneal cells in infected group were higher than those in control group(P < 0.05). In the infection group, the percentages of Th17 cells in spleen cells, peripheral blood leukocytes, and peritoneal cells were （1.31 ± 0.38）%, （1.85 ± 0.77）% and （2.90 ± 0.24）%, respectively, all significantly higher than those in the control group ［（0.59 ± 0.07）%, （0.35 ± 0.15）% and （0.41 ± 0.12）%, respectively］ (P < 0.05). Pearson correlation analysis showed that there was no correlation between percentages of Th17 and MDSCs in the spleen cells, peripheral blood leukocytes, and peritoneal cells in the infection group (r = -0.354, -0.746, 0.801; P > 0.05), while the monocytic MDSCs percentage was negatively correlated with Th17 cells in spleen in the infection group (r = -0.896, P < 0.05).  Conclusions The percentage of MDSCs and Th17 cells are both increased in mice at late stage of E. granulosus infection, and may play important roles in the development of echinococcosis.

Soluble expression, purification and bioinformatics analysis of β4-tubulin of Echinococcus granulosus protoscoleces

YAO Jia-qing, LIU Cong-shan, XUE Jian, TAO Yi, ZHANG Hao-bing*

2018, 36(3):  6-231-237. 

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Objective To analyze the β4-tubulin of Echinococcus granulosus protoscoleces （EgTub4） using bioinformatics tools, and optimize the induction of its soluble expression in Escherichia coli and purify the target protein. Methods The physicochemical properties and structure of EgTub4 protein were analyzed by ProtParam, SMART, Predictprotein and Swiss-model softwares. The signal peptide of EgTub4 sequence was predicted by the online software SignalP 4.1. Total RNA was extracted from E. granulosus protoscoleces and reversely transcribed into cDNA, based on which the EgTub4 gene was amplified by PCR. The recombinant prokaryotic expression vectors pET28a-EgTub4, pET30a-EgTub4 and pET32a-EgTub4 were constructed to explore the optimal induction conditions (including the expression vector, induction temperature, concentration of the inducer isopropyl-β-D-thiogalactoside, induction period, and medium composition) for soluble expression of the recombinant rEgTub4 protein. The recombinant protein was purified using nickel affinity chromatography, isolated by SDS-PAGE, and analyzed by Western blotting. 　Results  As predicted by bioinformatics analysis, the EgTub4 protein had a pI of 4.79 and a Mr of 49 700, did not contain any signal peptide, but had three domains: a GTPase domain, a C-terminal, and a coiled coil region. Double digestion and sequencing results confirmed the successful construction of the three recombinant plasmids(pET28a-EgTub4, pET30a-EgTub4 and pET32a-EgTub4). Western blotting analysis showed that the Mr values of recombinant protein in the expression vectors pET28a (+), pET30a (+) and pET32a (+) were 56 000, 60 000 and 68 000, respectively. The expression of EgTub4 gene in pET28a(+) was insoluble, mainly in the form of inclusion body, which needs to be purified under denaturing condition. The expression of EgTub4 gene in pET30a (+) and pET32a (+) was partially soluble, which can be purified under native conditions. In this study, pET30a (+) was selected as the expression vector of EgTub4 gene. The optimum induction conditions for soluble expression of target protein were: T = 25 ℃, [IPTG] = 0.01 mmol/L, t = 10 h, 2 × YT. The optimum concentration of imidazole for removing other protein was 150 mmol/L. The best elution concentration of imidazole was 500 mmol/L. Western blotting showed that the recombinant protein rEgTub4 could be specifically recognized by His-Tag and β-tubulin monoclonal antibody, resulting in a specific band at the Mr been 60 000.  Conclusion This study provides comprehensive bioinformatics prediction and analysis of EgTub4 which has been cloned, expressed and purified. 
  

Inhibitory efficacy of tacrine on Echinococcus multilocularis

LIU Cong-shan1, YIN Jian-hai1, YAO Jia-qing1, ZHANG Jing2, ZHANG Hao-bing1*

2018, 36(3):  7-238-245. 

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Objective To evaluate the effects of tacrine on Echinococcus multilocularis in vitro and against E. multilocularis infection in mice. Methods Tacrine was added at 100.00, 50.00, 25.00, 12.50 and 6.25 μmol/L to each well of a 96-well plate containing 50-200 E. multilocularis protoscoleces. The methylene blue exclusion method was used to determine the viabilities of protoscoleces on days 1, 3, 5 and 7 after the addition. The cytotoxicity of tacrine on three noncarcinogenic cell types(L929 cells, HK-2 cells and Chang liver) and three tumor cell types(A172, A2058 and HCT-8 cells) was tested using the Cell Counting Kit-8（CCK-8） method. In another experiment, 40 BALB/c mice with 6 months of E. multilocularis infection were randomly divided into 4 groups: 30 mg/kg tacrine group, 15 mg/kg tacrine group, 25 mg/kg mebendazole group, and 1% tragacanth group as a control. All the mice received intragastric administration for 28 days. After 14 days of withdrawal, the mice were sacrificed, and cysts in the peritoneal cavity were isolated and weighed to calculate the reduction rate of cyst weight. Data were analyzed with the nonparametric test in SPSS 17.0.  Results In vitro, tacrine showed an obvious dose-dependent effect on E. multilocularis protoscoleces. The viability of protoscoleces decreased with increased drug concentration and incubation time. After 3 days of exposure to 100 μmol/L tacrine, all the protoscoleces were dead, with dramatic morphological alterations. Intact parasites were hardly seen. After 7 days of tarcine treatment, the percentage dead of protoscoleces was (100 ± 0)%, (91.2 ± 2.5)%, (80.3 ± 5.1)%, (71.6 ± 2.4)% and (51.7 ± 2.9)% in the 100.00, 50.00, 25.00, 12.50, and 6.25 μmol/L groups, respectively. The normal host cell types were less affected by tarcine at 250.0 μmol/L, as demonstrated by the (27.6 ± 4.7)%, (29.6 ± 3.9)% and (26.9 ± 2.1)% inhibitory rate for L929 cells, HK-2 cells, and Chang liver cells, respectively. In contrast, tarcine exerted high cytotoxicity to tumor cells, as demonstrated by Tox50 values of (178.2 ± 3.2), (133.2 ± 5.2) and (128.8 ± 4.0) μmol/L for A172 cells, A2058 cells and HCT-8 cells, respectively. The reduction rate of cyst weight was -3.4%, 9.4%, and 38.2% in 30 mg/kg tacrine, 15 mg/kg tacrine and 25 mg/kg mebendazole groups, respectively. However, none was significantly different from the 1% tragacanthin group(P > 0.05). During the experimental period, 3, 6, 2 and 5 mice died in the four groups, and 25 mg/kg mebendazole and 15 mg/kg tacrine increased the survival rate compared with other groups.  Conclusion Tacrine can inhibit the activities of E. multilocularis protocoleces in vitro and increase the survival rate of mice infected with E. multilocularis.

Mass spectrometry identification and bioinformatics analysis of soluble proteins of Babesia microti

CAI Yu-chun, CHEN Shao-hong, LI Hao, LU Yan, AI Lin, CHU Yan-hong,

2018, 36(3):  8-246-252. 

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Objective To analyze the soluble proteins of Babesia microti by using proteomics and bioinformatics methods. Methods BALB/c mice were intraperitoneally injected with 100 μl of blood containing B. microti parasites to establish the mouse model of B. microti infection. Red blood cells were separated from the B. microti-containing blood of the infected mice at the peak of infection. The B. microti parasites were enriched by Percoll density gradient centrifugation, freeze-thawed and sonicated to obtain soluble proteins of B. microti. SDS-PAGE was used to identify the molecular weight distribution of proteins. The gel containing soluble proteins was divided into 2 samples by molecular weight and identified by the electrospray ionization mass spectrometry (ESI-MS) method. The ESI-MS data were collected. Protein data for Babesia spp., B. microti (strain RI) and Plasmodium falciparum were searched on the Uniprot KB database. The identified proteins were aligned with the protein sequences in the NCBI nr database, and Gene Ontology(GO) function associated with the aligned sequences of all identified proteins were extracted with the Mapping function in Blast2GO (Version 2.8.0). The GO annotations were used for analysis of biological processes, molecular functions, and cellular components of the identified proteins. The target protein sequence was aligned with the Babesia protein sequence in the KEGG GENES database using KAAS, and the relevant KEGG pathway was annotated with the KO number of the homologous/similar protein.  Results The B. microti parasites were enriched by Percoll gradient centrifugation and soluble proteins were obtained by freeze-thawing and sonication. SDS-PAGE showed that there were 5 major bands and 7 minor bands. After ESI-MS and alignment with proteins of Babesia spp., B. microti (strain RI) and P. falciparum, 757, 600 and 138 proteins were identified, respectively, of which 368, 375 and 12 had more than 2 unique peptide segments, respectively. Further analysis revealed that those with more unique peptide segments were surface antigens or secreted antigenic proteins, proteases, heat shock family proteins and rod-like proteins. Bioinformatics analysis resulted in 876 annotations for the soluble proteins according to the biological processes in which they are involved, 219 annotations by molecular function and 146 annotations by cell components. By KEGG annotation of homologous/similar proteins, a total of 172 KEGG signal/metabolic pathways related to the soluble protein sequences were extracted.  Conclusion The soluble proteins of B. microti are mainly composed of secretory proteins, proteases, and HSP family member proteins, as revealed by ESI-MS identification and bioinformatics analysis.

Cloning, expression of the gene for proteasome α5 subunit of Angiostrongylus cantonensis and the effect of the recombinant protein on human THP-1 macrophages apoptosis

YAN Lan-zhu1,2, SHI Xiao-meng1, ZU Yan-wen1, CHEN Xi-xi3,

2018, 36(3):  9-253-257. 

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Objective To obtain the gene for proteasome α5 subunit (pas-5) of Angiostrongylus cantonensis, construct the recombinant plasmid, and investigate the effect of PAS-5 recombinant protein on the apoptosis of human THP-1 macrophages. Methods The pas-5 gene was cloned from the cDNA of A. cantonensis, inserted into the prokaryotic expression vector pcoldⅢ to construct the recombinant plasmid pcoldⅢ-pas-5, and transformed into Escherichia coli DH5α competent cells. The cultures underwent PCR, double digestion, and sequencing to confirm the construction of recombinant plasmid. Expression of the recombinant plasmid was induced by IPTG, and the expressed recombinant proteins were analyzed by SDS-PAGE and Western blotting, and purified with Nickel affinity chromatography using Ni-NTA beads. The phorbol myristate acetate-induced THP-1 macrophages were incubated with PAS-5 for 18 h, and then flow cytometry was performed to evaluate the apoptosis of macrophages. The level of cytokine IL-10 in the culture supernatant was assessed by ELISA. Negative control and blank control groups were set with bovine serum albumin(BSA) and RPMI 1640, respectively, instead of PAS-5. Results The PCR amplification product of pas-5 was about 800 bp, consistent with expectation. The pcoldⅢ-pas-5 recombinant plasmid was verified by PCR, double digestion and sequencing results. SDS-PAGE results showed that the recombinant protein PAS-5 was expressed mainly in the form of inclusion body, with a relative molecular weight of 28 000. Western blotting showed that the recombinant protein could specifically bind to rabbit anti-mouse His monoclonal antibody. A single strip was obtained from Nickel affinity chromatography. The flow cytometry showed that the THP-1 macrophages apoptosis rate was(0.380 ± 0.194)% in the BSA group, and (0.052 ± 0.036)% in the PAS-5 group (P < 0.05). ELISA results showed a significantly higher level of supernatant IL-10 in the PAS-5 group than in the BSA group［(77.606 ± 1.766) pg/ml versus(45.652 ± 5.975) pg/ml, P < 0.05］.  Conclusion The recombinant plasmid pcoldⅢ-pas-5 was constructed, and the recombinant protein PAS-5 can inhibit the apoptosis of THP-1 macrophages, which may be related to the increased level of IL-10.
  

Establishment of developmental phenotype evaluation technique for adoptive RNAi schistosomula transfer

LIU Jian-fa1, LI Jian2, REN Yi-jing3, ZHANG Rui-xiang2, FANG Fang3, HU Wei2,4*

2018, 36(3):  10-258-265. 

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Objective To establish an approach for evaluating the development of Schistosoma japonicum receiving RNA interference, after being transferred to mice, in order to provide a tool for studying functional genes in the post-genomics era. Methods The primers of S. japonicum cathepsin B1(SjCB1) and green fluorescent protein(GFP) genes were designed to amplify  dsRNA. The 14-day-old schistosomula were collected from infected mice, and cultured by in vitro soaking with 6 mg/L SjCB1 dsRNA to specifically knock down the SjCB1 gene (SjCB1 interference group). In the control group, 6 mg/L green fluorescent protein (GFP) dsRNA was used instead (GFP interference group). The schistosomula after interference were surgically transferred back to the mouse mesenteric vein (3 mice in each group, 40 schistosomula/mouse). The adult worms were then collected after growing to sexual maturity. The numbers of male and female worms recovered, the pairing rate, the recovery rate, and the body length were analyzed. The growth and development of the adult worms were assessed. The relative expression level of SjCB1 gene in 14-day schistosomula and in recovered adult worms was detected by real-time PCR.  Results The recovered numbers of female and male worms, the pairing rate and the recovery rate in the SjCB1 interference group were (9.0 ± 1.4), (13.0 ± 2.9), 100% and 58.60%， and those in the GFP interference group were (9.0 ± 2.1), (11.3 ± 2.5), 100% and 54.67%, respectively. None was significantly different between the two groups (P > 0.05). The relative expression levels of SjCB1 in male and female schistosomula before adoptively transferred were 1.022 ± 0.019 and 0.643 ± 0.105, respectively, in the SjCB1 interference group, and both were significantly lower than those in the GFP interference group (2.880 ± 0.297 and 2.753 ± 0.164) (t = 0.000, 0.000; P < 0.01). The relative expression levels of SjCB1 in female and male worms recovered from the SjCB1 interference group were 1.286 ± 0.211 and 1.182 ± 0.287, respectively, which were significantly lower than those in the GFP interference group (5.508 ± 0.326 and 4.896 ± 1.230) (t = 0.013, 0.000; P < 0.01). The lengths of females in the SjCB1 interference group and the GFP interference group were (7.059 ± 1.255) and (8.433 ± 0.749) cm (t = 0.003, P < 0.01), and the lengths of males were (6.993 ± 2.734) and (10.561 ± 1.375) cm, respectively (t = 0.001, P < 0.01). There were no differences in the appearance, the internal reproductive system and the digestive system between the SjCB1 interference group and the GFP interference group. Females in both groups had eggs with a clear shape in the egg molds and the uterus.  Conclusion A standard procedure has been established to observe the developmental phenotypes in vivo of S. japonicum with RNA interference, and the SjCB1 gene has been knocked down by a soaking method. This procedure can be used to study the phenotypes of development-related genes.

Construction of the RSepitope-HPV16L1 fusion protein using the dominant epitope of ROP2-SAG1 antigen from Toxoplasma gondii and late structural protein 1 of HPV type 16 and #br# its expression in eukaryotic cells

XIE Zi-xin1, JIANG Jie2, WANG Wen-huan3, LV Jin-hui2, FENG Fang-fang2,

2018, 36(3):  11-266-274. 

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 Objective To construct the RSepitope-HPV16L1 fusion protein using the dominant epitope of ROP2-SAG1 antigen from Toxoplasma gondii (RSepitope) and late structural protein 1 of HPV type 16 (HPV16L1), and verify its expression in eukaryotic cells. Methods The recombinant plasmid pcDNA3.1/RSepitope was constructed after optimizing the RSepitope gene sequence. The gene fragment of HPV16L1 was amplified from the plasmid T-HPV16L1 preserved in our lab, and the recombinant plasmid pcDNA3.1/RSepitope-HPV16L1 was constructed. The recombinant plasmid was verified by enzymatic digestion, PCR amplification and sequencing, and transfected into the COS-7 cells. After 48 h of culture, cells were collected to extract RNA. RT-PCR was performed to amplify target genes. Meanwhile, Western blotting and indirect immunofluorescence test were performed to detect the expression of target proteins RSepitope and RSepitope-HPV16L1 in cells.  Results After amplification and enzymatic digestion, a 282-bp fragment was obtained from pcDNA3.1/RSepitope, and a 1 500 bp fragment was obtained from pcDNA3.1/RSepitope-HPV16L1. RT-PCR using cDNA of the recombinant plasmid-transfected cells as the template produced specific bands of 282 bp and 1 500 bp, respectively. Western blotting analysis showed that when the RSepitope anti-serum was used as the primary antibody, specific bands with relative Mr of 10 000 and 65 000 were seen for RSepitope and RSepitope-HPV16L1, respectively.  When anti-HPV16L1 monoclonal antibody was used as the primary antibody, a specific band with relative Mr of 65 000 was seen for RSepitope-HPV16L1. Indirect immunofluorescence analysis using RSepitope anti-serum as the primary antibody produced green fluorescence in cells transfected with pcDNA3.1/RSepitope and pcDNA3.1/RSepitope-HPV16L1. When the HPV16L1 monoclonal antibody was used as the primary antibody, green fluorescence was seen only in cells transfected with pcDNA3.1/RSepitope-HPV16L1.  Conclusion The RSepitope-HPV16L1 fusion is constructed using RSepitope and HPV16L1 and could be expressed in eukaryotic cells.

Recombinant expression of calcium-binding protein from Clonorchis sinensis and preliminary evaluation of its application in immunodiagnosis

PU Jue-biao1,2，TANG Li-li3，YIN Cen-nan1，DU Xin-yue1, WU Jian-hua1, ZHAO Wei1,

2018, 36(3):  12-275-279. 

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Objective To clone, express the calcium-binding EF-hand-domain-containing protein of Clonorchis sinensis (Cs16) by using the prokaryotic and eukaryotic expression systems, and evaluate its application in immunodiagnosis. Methods The Cs16 gene was amplified by PCR using specific primers and cDNA library of Clonorchis sinensis, and sub-cloned into prokaryotic and eukaryotic expression vectors pGEX-4T-1 and pPIC9K, respectively. The recombinant vectors pGEX-4T-1-Cs16 and pPIC9K-Cs16 were transformed into Escherichia coli BL21 and yeast GS115 for protein expression. The recombinant proteins were purified and analyzed for purity and antigenicity by SDS-PAGE and Western blotting, respectively. The purified recombinant protein was used as the coating antigen for indirect ELISA to evaluate its diagnostic performance against serum samples from 24 clonorchiosis patients and its cross-reactivity to serum samples from schistosomiasis patients.  Results PCR resulted in a 515 bp of Cs16 fragment. The recombinant plasmid pGEX-4t-1-Cs16 and pPIC9K-Cs16 were verified by enzymatic digestion and sequencing. SDS-PAGE analysis showed that the two recombinant plasmids were solubly expressed in E. coli BL21 and yeast GS115, producing recombinant protein Cs16 with Mr of 16 000. Western blotting results showed that the recombinant proteins were specifically recognized by anti-GST and anti-His. ELISA showed that the sensitivity of recombinant Cs16 proteins expressed in the prokaryotic and eukaryotic expression systems was 70.8%(17/24) and 54.2%(13/24), respectively, and the specificity was 95.2% (20/21) and 100% (21/21), respectively, with significant differences between the two systems (P　<　0.05). The cross-reactivity with sera from schistosomiasis patients was 1/10.  Conclusion The recombinant Cs16 can be expressed prokaryotically and eukaryotically, and has potential applications in immunodiagnosis of clonorchiasis.
  

Analysis of current status of human intestinal protozoal infection in Henan Province

LIU Ying1, LI Su-hua1, ZHANG Ya-lan1, LIU Ruo-ning2, QIAN Dan1, YANG Cheng-yun1,ZHOU Rui-min1, HE Li-jun1, LU De-ling1, DENG Yan1, CHEN Wei-qi1, ZHAO Yu-ling1, ZHANG Hong-wei1, XU Bian-li1*

2018, 36(3):  13-280-285. 

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 Objective To understand the current status of human intestinal protozoal infection in Henan Province. Methods An epidemiological survey was carried out in Henan Province according to the Protocol of National Survey Scheme on Major Human Parasitic Diseases, in counties selected based on the ecological and economic conditions. A stratified sampling method was used to select one village in each county, and 250 residents in each village were examined. Fresh human fecal samples were collected, in which trophozoites and/or cysts of intestinal protozoa were examined by physiological saline direct smear method and iodine stain method, respectively. The infection rate was compared between different populations using the chi-square test. 　Results  A total of 26 866 persons from 104 villages in 35 counties(cities) were examined, and the overall infection rate of intestinal protozoa was 0.57%(152/26 866). Seven species of intestinal protozoa were detected: Iodamoeba buetschlii, Entamoeba hartmani, Endolimax nana, Entamoeba histolytica, Entamoeba coli, Giardia lamblia and Blastocystis hominis, of which the Endolimax nana conferred the highest infection rate (0.19%, 50/26 866). There was a significant difference in infection rate among the species (P < 0.05). Among the 4 ecological regions, the North China Plain Ecological Region had higher infection rate (0.67%, 45/6 696), with no significant difference (P > 0.05). After normalization, Xiangcheng had the highest infection rate among the 27 counties (cities) detected with human intestinal protozoa (2.69%). The infection rate was higher in males (0.73%, 95/12 965) than in females (0.41%, 57/13 901) (P < 0.05). Among different age groups, the infection rate was higher in the groups of < 20 (0.66%, 31/4 667) and 20- (0.66%, 18/2 742), predominated by Blastocystis hominis. Furthermore, among different occupations, infection rate in workers and farmers was 0.76% (4/528) and 0.54% (96/17 896), respectively. Populations with a level of high school or above and a primary school showed an infection rate of 0.71% (18/2 550) and 0.52% (42/8 014), respectively. The Han ethnicity had a higher infection rate (0.57%, 151/26 434) (P < 0.05). The average annual income per farmer in the survey was 1 200-23 590 yuan, with a median of 5 950 yuan. There was no significant difference in the intestinal protozoa infection rate in age, occupation, education level, or income (P > 0.05).  Conclusion Seven species of intestinal protozoa are identified with a low rate of infection in Henan Province.

Supervision and evaluation of malaria elimination in China: A political, economic, social and technological analysis

SU Jie1,2, LI Hong-mei1, DING Wei1, MA Xue-jiao1, HUANG Lu-lu1, WANG Ru-bo1, GUAN Ya-yi1*

2018, 36(3):  14-286-290. 

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In this study, a PEST(political, economic, social and technological) analysis was performed on supervision and evaluation of malaria elimination program in China, in order to look into the the political, economic, social, and technological factors during the process. The objective of this work was to provide a scientific basis for improving the effectiveness of supervision and evaluation of malaria elimination.
  

Current status of echinococcosis control in China and the existing challenges

WANG Tian-ping*, CAO Zhi-guo

2018, 36(3):  15-291-296. 

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This review summarizes the results of epidemiological survey and thematic surveys on echinococcosis, as well as the endemic situation of echinococcosis in China. The implementation of echinococcosis control programs, strategies, and technical problems in prevention and control of the disease are also reviewed.

Research progress on drug treatment for hydatidosis

XU Shuo1, DANG Zhi-sheng2, ZHANG Hao-bing2, HU Wei2, ZHAO Yu-min1*

2018, 36(3):  16-297-302. 

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Hydatidosis is a serious zoonotic parasitic disease caused by Echinococcus larvae within the human body. The disease distributes all over the world except the Antarctica region, significantly affecting the development of animal husbandry and impairing human health. As one of the therapies for human hydatidosis, drug treatment shows promising clinical efficacy. Development on drug treatments has been made due to the accumulating research and treatment experience. This paper reviews the research progress on the treatment of the hydatidosis with some western medicine and Chinese traditional medicine.

Research development on neurotransmitters involved in Biomphalaria infection with Schistosoma mansoni

HUANG Tao, GUO Yun-hai, ZHANG Yi*

2018, 36(3):  17-303-306. 

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Schistosoma mansoni poses a serious threat to human health and has been identified as an important parasite that undergoes a sexual developmental phase within a planorbid snail host. Currently, research on Biomphalaria infection with S. mansoni is under a switch from the macro-scale to micro-scale, with a large number of functional genes and proteins being reported. Neurotransmitters are important substances that regulate nerve activities and are widely distributed within both Biomphalaria snails and S. mansoni. Their activity is not limited to the regulation of basic physiological behaviors of hosts, but also exerts influences on the snail’s oviposition and plays a regulatory role in the development of the parasite. Studies have shown that serotonin, dopamine, histamine, acetylcholine, glutamate and nitric oxide are the most common neurotransmitters involved in S. mansoni infection. This paper provides insights into the physiology, biochemistry and tissue localization of these neurotransmitters and also comprehensively provides evidence for the compatibility between Biomphalaria snails and S. mansoni.

Research progress on the role of IL-33/ST2 axis in infectious diseases

JIANG Chun-jie, GUAN Fei, LEI Jia-hui*

2018, 36(3):  18-307-314. 

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The interleukin-33(IL-33) is a IL-1 family cytokine newly discovered in 2005, and it functions by specifically binding to its receptor suppression of tumorigenicity 2(ST2), forming the IL-33/ST2 axis. The IL-33/ST2 axis plays different roles in different infectious diseases, depending on the type of infectious agents, organ involved, infection stage (acute or chronic), and cellular and cytokine microenvironments. In this review, we focus on recent research progress on diverse roles of the IL-33/ST2 axis in various infectious diseases caused by bacteria, viruses, fungi and parasites.

A KAP survey on taeniasis and cysticercosis in Fangcheng County of Henan Province in 2016

DENG Yan1, ZHANG Ya-lan1, LI Su-hua1, CHEN Wei-qi1, LIN Xi-meng1, CHEN Xi2, WANG Ting-zhu3, LI Peng1, ZHANG Hong-wei1, XU bian-li1*

2018, 36(3):  19-242-245. 

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 Six towns in Fangcheng County, where taeniasis and cysticercosis cases were reported, were selected as the survey spots in 2016, accompanied by employment of towns with intervention (Yangji, Bowang and Dushu towns) or control (Erlangmiao, Yanglou and Xiaoshidian towns). The KAP survey on taeniasis/cysticercosis was carried out in the manner of structured questionnairing, and results were analyzed by sex, age, and occupation using the SPSS 22.0 software. The total awareness rate of taeniasis/cysticercosis knowledge was 0.5% (13/2 399), and there was no statistically significant difference between the intervention (0.6%, 7/1 128) and control towns(0.5%, 6/1 271) (χ2 = 0.65， P > 0.05). The awareness rates in males and females were 1.0% (11/1 179) and 0.2% (2/1 320), respectively (χ2 = 8.30, P < 0.01). Among the five questions in the questionnaire, the question regarding knowledge on the correlation of cysticercus cellulosae-containing pork with diseases had the highest score rate (61.3%, 1 471/2 399), followed by the infection route of taeniasis (1.3%, 32/2 399), common symptoms of cysticercosis (0.8%, 20/2 399) and taenia basic morphology (0.6%, 15/2 399), then the infection route of cysticercosis (0.3%, 7/2 399). The percentage of residents who had eaten cysticercus-containing pork was 7.9% (61/774) in the age group of > 60, significantly higher than that in the group of < 60 (2.2%, 36/1 625) (χ2 = 43.38, P < 0.01). And the percentage of those who once raised pigs was 33.5% (82/245), significantly higher than that of those who did not (0.7%, 15/2 154) (χ2 = 608.97, P < 0.01). The proportion of persons who had an eating habit for uncooked meat or incompletely-cooked meat was 20.3% (488/2 399). Of them 94.9% (463/488) used to taste dumpling stuffing containing raw meat, and the females accounted for 25.3% (334/1 320), significantly more than the males (12.0%, 129/1 079) (χ2 = 67.91, P < 0.01). Only 3.5% (85/2 399) of the residents would completely separate the raw and the cooked meat during cooking.  Tasting dumpling stuffing and mixing the raw and the cooked food together are the main risk behaviors in local residents. It is important to strengthen health education on taeniasis/cysticercosis.

Epidemiological investigation of hydatid disease prevalence among students in Yushu Prefecture of Qinghai Province

CHENG Shi-lei, MA Xiao*, CAI Hui-xia, LIU Na, LIU Yu-fang, SHI Ke-mei

2018, 36(3):  20-263-265. 

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To understand the prevalence of hydatid disease among students in Yushu Prefecture, 22 schools were randomly selected from 16 towns in 6 counties including Chengduo County, Nangqian County, Qumalai County, Yushu County, Zaduo County and Zhiduo County from June to August, 2012. Students aged 7-15 were examined by B-ultrasound and serum ELISA. A total of 7 454 students were examined by B-ultrasound, of whom 58 were detected with hydatid cysts, with a prevalence of 0.78%. A total of 7 081 students were examined by ELISA, of whom 302 were detected to be positive for anti-Echinococcus antibody, with a positive rate of 4.26%. The detection rates by B ultrasound in males and females were both 0.78% (30/3 853, 28/3 601). The antibody positive rate in females was 4.62% (158/3 423), and 3.94% in males (144/3 658) (P ＞ 0.05). Among different regions, the Chengduo County had the highest B ultrasound detection rate (1.67%, 27/1 612) (χ2 = 35.224, P < 0.05), while Qumalai had the highest positive rate of anti-Echinococcus (6.68%, 82/1 228) (χ2 = 32.817, P < 0.05). Among the different age groups, the group of >14 years ranked top with regard to the detection rate by B ultrasound (1.50%, 10/666)(χ2 = 13.451, P < 0.05) and the positive rate of anti-Echinococcus (7.20%, 46/639) (χ2 = 53.284, P < 0.05). The results suggested a high prevalence and a high positive rate of anti-Echinococcus among students in Yushu Prefecture of Qinghai Province. Efforts should be made to strengthen the education and surveillance of hydatid disease among students.
  

Identification of Leishmania in Shaanxi Province based on repeated DNA sequence

LI Xin-yan1,2, ZHANG Zheng1, LIU Dong-li1*, WANG Feng-ping3, WANG An-li1, WANG Tian-hai3, SHI Yi1, GUAN Rong-hui1, LI Wen-juan1

2018, 36(3):  21-271-274. 

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In this study, PCR was performed to amplify repeated DNA sequence in Leishmania DNA extracted from a sample of Leishmania isolate from dog, a bone marrow sample from an infected dog, 3 bone marrow samples from patients, and two Phlebotomus samples. The amplified products underwent bi-directional sequencing after capillary electrophoresis. The DNAstar SeqMan software was used for sequence assembly, and the sequences were aligned with other DNA repeated sequences reported in literature. Homology was analyzed and phylogenetic trees were constructed using the neighbor joining method. PCR resulted in a product of ~260 bp for all the 7 samples, which was consistent with expectation and verified by sequencing. Results of sequence alignment showed that the products from the 7 samples differed from the reference sequences of L. infantum (GenBank No. L42486) and L. donovani (GenBank No. L42486) in only 1 base. Homology analysis revealed over 90% species homology to the reference sequences, with some samples reaching 100%. The phylogenetic tree showed that the 7 sequences clustered into a branch with L. infantum and L. donovani. Combined with the epidemiological data, these results suggest that the Leishmania species in Shaanxi Province is L. infantum.
  

Observation of hemocytes of Oncomelania hupensis by acridine orange staining

WEI Xiang-hong1, 2, TAN Ping1, 2*, WAN Lun3, LU Ben4, ZHENG Mei-ling4, WU Zhi-ling4, JIANG Yuan4

2018, 36(3):  22-315-317. 

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 Hemolymph smears were prepared after extruding soft tissues of Oncomelania hupensis and stained by fluorescent dye acridine orange. The hemocytes were observed under a fluorescence microscope. The following 12 types of hemocytes were clearly seen, including round hemocytes with abundance of red cytoplasm, round hemocytes with abundance of red cytoplasm and intracellular granules, round hemocytes with little red cytoplasm, round hemocytes with little red cytoplasm but the presence of intracellular granules, round hemocytes with abundance of green cytoplasm, round hemocytes with abundance of green cytoplasm and intracellular granules, round hemocytes with little green cytoplasm, round hemocytes with little green cytoplasm but the presence of intracellular granules, hemocytes with abundance of cytoplasm and pseudopodia, hemocytes with little cytoplasm but the presence of pseudopodia, fusiform hemocytes with red cytoplasm, and fusiform hemocytes with green cytoplasm. The results indicated that the method could be used for the staining of hemocytes of O. hupensis, which will provide technical support for studies on shellfish immune defense and the effects of various biological, physical and chemical factors on hemocytes of O. hupensis.