Table of Content

30 April 2019, Volume 37 Issue 2

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ORGINAL ARTICLES

Effect of artesunate on expression of heat shock protein 47 in mice with liver fibrosis induced by Schistosoma japonicum

Yong-hua ZHOU, Chen XU, Ying-ying YANG, Chun-gang ZHOU, Cong-jin MEI, Xue SAI, Yong-liang XU, Jun-qi YANG, Li-juan SHEN

2019, 37(2):  115-121.  doi:10.12140/j.issn.1000-7423.2019.02.001

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Objective To understand the possible mechanism underlying the artesunate’s inhibition on Schistosoma japonicum-induced hepatic fibrosis, the effect of artesunate on the expression of heat shock protein 47 (HSP47) in serum and liver during the early stage of mouse hepatic fibrosis induced by S. japonicum infection was studied. Methods Total 30 female ICR mice were randomly divided into 3 groups: the un-infected control group, the infection group and the treatment group (infection + artesunate treatment), with 10 mice in each group. Each mouse in the infection and treatment groups was infected with 20 ± 1 cercariae of S. japonicum via abdomen skin penetration. All infected mice were treated with 300 mg/kg praziquantel for 2 days to eliminate the adult worms and the mice in treatment group were additionally treated with 60 mg/kg artesunate in 0.3 ml volume by intraperitoneal injection daily for 2 weeks. For the control group, mice were given the same volume of saline. After the artesunate treatment, all mice from 3 groups were sacrificed, blood samples were taken for serum separation, and the levels of HSP47 and transforming growth factor-β1(TGF-β1) in sera were detected by ELISA. The mouse livers and spleens were collected and weighed to calculate their organ index(organ weight/body weight). The livers were fixed with paraformaldehyde and sectioned for hematoxylin eosin (HE) and Masson-trichrome staining to observe the liver pathological changes and collagen proliferation. The hydroxyl-proline level in liver was measured using the alkaline hydrolysis. The mRNA expression levels of liver HSP47 and type Ⅰ collagen (COL1) were determined by real-time quantitative PCR (RT-PCR). Results The liver index in infection group (10.50 ± 0.57) was significantly higher than that in treatment group (8.31 ± 0.52) and in un-infected control group (4.72 ± 0.52) with statistical difference (P < 0.01). The spleen index was significantly higher in infection group (2.41 ± 0.44) than that in un-infected control group (0.38 ± 0.04) (P < 0.01), however it was not significantly different compared to that in treatment group(P > 0.05). Liver pathological damage was significantly reduced in treatment group compared to the infection group, including reduced liver size and egg granuloma. Masson-trichrom staining showed less liver fibrosis in treatment group than in infection group. The area of collagen proliferation in liver in treatment group was (6.87 ± 3.54)%, that is significantly smaller than that in infection group [(25.78 ± 2.61)%] (P < 0.01). The levels of HSP47 and TGF-β1 in sera of mice in the treatment group [(169.81 ± 20.94) pg/ml and (20.82 ± 1.90) ng/ml, respectively] were significantly lower than that in infection group [(203.14 ± 46.29) pg/ml and (27.49 ± 6.81) ng/ml] (P < 0.05), even though the levels of HSP47 and TGF-β1 in both infection and treatment groups were significantly higher than that in the un-infected control group (P < 0.01). The similar results were also observed in the hydroxyproline level and the expression of HSP47 and COL1 in liver tissues. The level of liver hydroxyproline in the treatment group was (0.27 ± 0.08) mg/g, which was significantly lower than that of the infection group [(0.69 ± 0.07) mg/g] (P < 0.01), however, all higher than that in un-infected control group [(0.11 ± 0.04) mg/g] (P < 0.05). The mRNA expression levels of HSP47 and COL1 in the liver of mice of treatment group were 0.49 ± 0.27 and 0.67 ± 0.09, respectively, which are significantly lower than those in the infection group (0.84 ± 0.17 and 0.91 ± 0.11, respectively) (P < 0.05). The expression levels of HSP47 and COL1 in both infection and treatment groups were significantly higher than those in the un-infected control group (0.24 ± 0.04 and 0.24 ± 0.05) (P < 0.01). The serum HSP47 level was positively correlated with the level of TGF-β1 (r = 0.928 0), and serum HSP47 level and TGF-β1 level were positively correlated with the level of hepatic COL1 mRNA expression(r = 0.926 2, 0.872 8). Conclusion Artesunate has protective effects on the early hepatic fibrosis caused by S. japonicum infection and the protection is possibly achieved by down-regulating the expression of HSP47, further suppressing the collagen synthesis so as to alleviate the hepatic fibrosis. HSP47 is expected to be one of the new prognostic markers and treatment targets in S. japonicum infection.

Identification of Schistosoma japonicum schistosomula proteins interacted with Microtus fortis resistance-related ILKAP by co-immunoprecipitation

Rong LI, De-hui XIONG, Wei-xin HU

2019, 37(2):  121-126.  doi:10.12140/j.issn.1000-7423.2019.02.002

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Objective To understand the mechanism underlying the natural resistance to Schistosoma infection in Microtus fortis, the schistosomula proteins interacted with ILKAP(integrin-linked kinase-associated serine/threonine phosphatase), one of the resistant-related proteins in M. fortis, were screened and identified. Methods The crude proteins were extracted from S. japonicum schistosomula and those proteins specifically bound with M. fortis ILKAP(MfILKAP) were isolated and identified by immunoprecipitation using magnetic beads conjugated with anti-ILKAP IgG. Briefly, the beads conjugated with anti-ILKAP IgG were co-incubated with bone marrow proteins of M. fortis and S. japonicum schistosomula extracts, the proteins bound to the beads were extracted in SDS-loading buffer and separated by SDS-PAGE. The separated bands were cut from the gel and analyzed by MALDI-TOF-MS. The schistosomula proteins bound to ILKAP were identified by searching through genome database of S. japonicum. In order to further determine the binding activity, genes for one of the ILKAP-bound S. japonicum proteins, tegument antigen （TA), and MfILKAP were amplified and cloned into eukaryotic expression plasmids Flag-pCMV-2b and Myc-pcDNA3.1/(-)b, respectively. The recombinant plasmid DNAs were co-transfected into HEK293T cells. The expression of recombinant tegument antigen of S. japonicum (SjTA) and MfILKAP and their interaction were confirmed by co-immunoprecipitation with anti-ILKAP. Results A total of 8 S. japonicum schistosomula proteins bound to MfILKAP were identified by co-immunoprecipitation with anti-ILKAP IgG and MALDI-TOF-MS analysis. One of the major ILKAP-binding proteins was SjTA. After RT-PCR amplification and enzymatic digestion, fragments of 1 147 bp, and 4 300 bp from M. fortis, and fragments of 561 bp and 5 500 bp from Schistosomula were obtained, respectively. In order to further determine their interacting activity, MfILKAP and SjTA were successfully cloned from their corresponding cDNA and the recombinant proteins were effectively expressed in HEK293T cells determined by Western blotting with specific antibodies. The binding activity of recombinant MfILKAP to recombinant SjTA was further confirmed by co-immunoprecipitation with anti-ILKAP IgG from the co-expressed cell lysates and recognition by the specific antibodies. Conclusion Total 8 S. japonicum schistosomula proteins have been identified to bind to MfILKAP. One of the ILKAP-binding proteins, SjTA, was cloned and co-expressed in HEK293T cells. The interaction between SjTA and MfILKAP was further confirmed by the binding of both recombinant proteins.

Schistosoma japonicum soluble worm proteins and recombinant cystatin ameliorate experimental colitis in a murine model

Yi WU, Lu LI, Yong-wei XU, Rui-xin XING, Jing HU, Shu-shu WANG, Ji-long SHEN, Yuan-hong XU, Xi CHEN

2019, 37(2):  127-136.  doi:10.12140/j.issn.1000-7423.2019.02.003

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Objective To explore the potential therapeutic effect of Schistosoma japonicum soluble worm protein (SjSWP) and recombinant cystatin (rSjcystatin) on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in a mouse model that mimics human inflammatory bowel diseases(IBDs). Methods Twenty-four female BALB/c mice were divided into four groups: non-treated control group(control), phosphate buffer (PBS) treated group, rSjcystatin treated group (rSjcystatin) and SjSWP treated group(SjSWP). The PBS, rSjcystatin, SjSWP treated groups were respectively intraperitoneally injected with 100 μl PBS, 50 μg rSjcystatin and 50 μg SjSWP at 6 h and 24 h after being induced with 0.1 ml of 5% TNBS by enema for colitis, while the control group was injected with the same amount of PBS at corresponding time without colitis induced. After 72 hours of TNBS enema, mice in each group were sacrificed under anesthesia. The pathological and inflammatory parameters were determined by measuring macroscopic score, myeloperoxidase (MPO) activity, colon length and microscopic score. The Th1 to Th2 cells and T regulatory cells (Tregs) in the spleen of mice were measured by flow cytometry, and the immune responses induced by rSjcystatin and SjSWP were investigated. The levels of cytokines including interferon-gamma (IFN-γ), interleukin-4 (IL-4), IL-13, IL-10 in the colon tissue of mice were detected by enzyme-linked immunosorbent assay (ELISA). Multiple groups of measurement data were analyzed by one-way analysis of variance, and pairwise comparisons between multiple groups of mean were tested by Student-Newman-Keuls. Results The mice with TNBS-induced colitis showed significant inflammation and pathology characterized by the weight loss, rectal bleeding, diarrhea, colon shrinking and ulceration, as well as induced inflammatory cytokine INF-γ, inflammatory cell infiltration and increased MPO activity. In this study we identified that treatment with rSjcystatin and SjSWP significantly reduced the TNBS-induced colon inflammation characterized by reduced disease manifestations in macroscopic score (improved colon erosion, adhesion, ulceration and colon shrinking) and microscopic scores (less tissue damage and inflammation), compared to the group of mice received PBS only. The macroscopic scores of colon in the control group, PBS group, rSjcystatin group and SjSWP group were 0, 9.8 ± 0.8, 4.3 ± 1.0 and 4.5 ± 1.4; the mean microscopic scores of mice were 0, 6.00 ± 0.4, 2.2 ± 0.5 and 2.8 ± 0.6, respectively. Among the above results, rSjcystatin and SjSWP had significant differences compared with PBS group (F = 57.8, 34.1, P < 0.01). There was no significant difference between rSjcystatin group and SjSWP group（P > 0.05）. MPO values in control group, PBS group, rSjcystatin group and SjSWP group were (0.3 ± 0.0), (1.4 ± 0.1), (0.6 ± 0.1) and （0.6 ± 0.1）（F = 32.5, P < 0.01）; the Tregs in spleen were (4.4 ± 0.7), (8.2 ± 3.1), (18.4 ± 2.1) and (13.4 ± 1.9)% （F = 8.3, P < 0.01); the IFN-γ and IL-10 in colon tissue homogenate supernatants of colon tissue were (952.0 ± 61.1), (1 684.6 ± 158.3), (1 092.0 ± 81.9) and (1 001.6 ± 111.2) pg/ml; (322.8 ± 27.4), (399.3 ± 77.9), (860.6 ± 153.6) and (892.0 ± 113.8) pg/ml respectively （F = 9.6, 8.3, P < 0.05). The results showed that, in rSjcystatin group and SjSWP group, the inflammatory cytokine INF-γ, MPO activity in colon tissue decreased, combined with boosted Tregs response and the secretion of regulatory cytokine IL-10, compared to the group of mice received PBS only with statistical difference (P < 0.01). There was no significant difference for the therapeutic efficacy on inflammatory colitis between rSjcystatin group and SjSWP group(P > 0.05). Conclusions SjSWP and rSjcystatin could alleviate experimental colitis in mice via stimulating Tregs and Th2 mediated responses and, correspondingly, suppressing Th1 pro-inflammatory response related to the pathogenesis of colitis.

Analysis of miRNA expression in Echinococcus granulosus protoscoleces isolated from sheep liver and lung

Shi-qiang FU, Hai-ning FAN, Hai-jiu WANG, Ying ZHOU, De-ping CAO, Yan-fei LI, Zhi-xin WANG, Li REN

2019, 37(2):  137-143.  doi:10.12140/j.issn.1000-7423.2019.02.004

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Objective The expression of microRNA (miRNA) in Echinococcus granulosus protoscoleces isolated from liver (hepatic protoscoleces, HPSC) and lung (lung protoscoleces, LPSC) of infected sheep was analyzed, and the specific miRNAs expressed in E. granulosus were identified by screening available databases in order to provide E. granulosus miRNA information for further study. Methods The fresh sheep livers and lungs with hydatid cysts were collected from a slaughter house near the suburb of Xining City. The hydatid cysts were extracted, filtered and precipitated to obtain pure protoscoleces of E. granulosus. The total RNA was extracted from cysts using TRIzol, and the HPSC and LPSC small RNA (sRNA) libraries were constructed. The deep sequencing was performed on these libraries using high-throughput sequencing technology. The obtained sequences were used to compare with databases including miRBase, Pre-miRNA database, RFam and Repbase to obtain E. granulosus specific miRNA information. The secondary structure of E. granulosus miRNA was predicted using mfold software. The differences in miRNA expression between HPSC and LPSC were compared. Results The E. granulosus HPSC and LPSC sRNA libraries were successfully constructed and total 10 182 508 and 11 133 735 effective sequences, respectively, were obtained. Most of the sequences were 18-22 nucleotide (nt) long, of which 22 nt had the highest frequency accounting for 20.9% (2 128 728/10 182 508) for HPSC and 24.6%(2 737 570/11 133 735) for LPSC, followed by 19 nt (14.0%, 1 429 846/10 182 508; 18.5%, 1 880 607/10 182 508) and 21 nt (16.9%, 1 875 698/11 133 735; 16.9%, 1 885 560/11 133 735), respectively. Except for 87 and 79 known miRNA, total 341 and 339 new miRNAs were identified in the constructed E. granulosus HPSC and LPSC sRNA libraries, respectively. Among them 58 for HPSC and 62 for LPSC were conserved with sequences of Schistosoma japonicum, S. mansoni, Platyhelminthes and Gyrodactylus. In the known miRNA of E. granulosus, 8 miRNA including egr-miR-36a-3p, egr-miR-36b-3p, egr-miR-10229a-5p, egr-miR-10233-5p, egr-miR-10240-3p, egr-miR-10243-5p, egr-mir-10252-5p and egr-mir-10287-5p were specifically expressed in the HPSC library, and the remaining miRNA were expressed in both HPSC and LPSC sRNA libraries at the same level. Among other helminth-conserved miRNA, 50 were co-expressed in both HPSC and LPSC, 8 were specifically expressed in HPSC and 12 in LPSC. Among the new identified miRNA, 78 were specifically expressed in HPSC and 76 in LPSC. Of the 574 miRNA identified in two libraries, 392 miRNA were co-expressed in both libraries with following top 10 highest expression miRNAs: egr-miR-1-5p, egr-miR-2a-3p, egr-miR-2c-3p, egr-miR-2b-3p, egr-miR-7-5p, egr-let-7-5p, egr-miR-7b-5p, egr-miR-9-5p, egr-miR-9-3p, egr-miR-10a-5p. Compared with HPSC, 124 miRNAs were up-regulated by more than 2-fold and 30 miRNAs down-regulated by 0.5-fold in LPSC. Among the total miRNA identified in both libraries, 94 and 88 were specifically expressed in HPSC and LPSC, respectively. Conclusion This study identified the specific expression of miRNA in HPSC and LPSC.

Proteomic analysis of cyst fluid of Cysticercus cellulosae

Jian-da PANG, Yi-ning SONG, Xin-rui WANG, Ming-yuan LIU, Shu-min SUN

2019, 37(2):  144-149.  doi:10.12140/j.issn.1000-7423.2019.02.005

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Objective The protein components of cyst fluid of Cysticercus cellulosae were analyzed by liquid chromatograph-mass spectrometer (LC-MS/MS) to identify the expression profile of C. cellulosae, The protein profile of Cysticercus fluid can be used to screen for the specific antigens as diagnostic markers and analyze the function of Cysticercus protein components involved in the invasion and survival of Cysticercus in the host. Methods The cystic fluid was aseptically collected from an infected pig and the protein components of the fluid was analyzed by LC-MS/MS after being digested with trypsin. The Blast2Go software was used for the comparison and annotation, and Gene Ontology (GO) was used to analyze classification of the annotated protein components. Results A total of 486 proteins were obtained by LC-MS/MS analysis, 158 of them were known proteins and the rest 328 were unknown proteins after being analyzed by searching with Mascot 2.2 software. The primary searching results identified that cystic fluid contains four highly abundant proteins (8 kDa glycoprotein, annexin, enolase, trypsin-like protein), and other specific antigens and a variety of enzymes, mainly located intracellularly and as cell components, of which annexin has binding functions mainly involved in a series of calcium-dependent membrane-type biological activities. The enolase has ionic binding function and hydrolase activity involved in the regulation of DNA-binding transcription factor and the glycolytic pathway. The results of GO analysis showed that 65 proteins were located intracellularly, 64 proteins located in the cell components, 25 proteins located in the organelles, and the rest located in the organelles, protein complexes, extracellular regions and cell membranes. In terms of molecular functions of identified proteins, 72 proteins have catalytic activities, 74 proteins have binding functions, and the remaining proteins are involved in transporter activities, molecular function regulators, structural molecular activities, etc. In terms of biological process classification, 69 proteins are involved in the cell process, 73 involved in the metabolic process, and 11 involved in the regulation of biological processes, signals, response to stimuli and cell localization. Conclusion The proteomic results of cyst fluid of Cysticercus identified a total of 486 known or unknown proteins, 4 of them are highly abundant proteins that can be used as candidate antigens for immunodiagnosis of cysticercosis. The identified enzymes may play important roles in the interaction between C. cellulosae and the host, providing a basis for further exploration of the invasion and parasitic mechanism of C. cellulosae.

Follow-up of a patient with Leishmania and human immunodeficiency virus co-infection who received prolonged sodium stibogluconate treatment

Shi-hui SONG, Xi-en GUI

2019, 37(2):  148-152. 

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Objective To observe the curative effect and prognosis of prolonged sodium stibogluconate treatment in a case of Leishmania and human immunodeficiency virus(HIV) co-infection. Methods Clinical information of the patient was collected. The curative effect and prognosis were followed-up. Results The patient was working in Jiuzhaigou County of Sichuan Province （an endemic area of visceral leishmaniasis） in 1990. At the end of 1992 he presented with fever, hepatomegaly and splenomegaly, and was diagnosed with liver cirrhosis by local hospitals. The symptoms were not improved after symptomatic treatment, with continued hapastosplenomegaly, accompanied by severe anemia and weight loss. He was later diagnosed with visceral leishmaniasis via bone marrow examination in 1994, and given intravenous infusions of sodium stibogluconate (pentavalent antimony 600 mg/d for 6 days). After 6 days of treatment, his condition got improved. In August 2010, an abdominal mass was found in the left upper quadrant, accompanied by abdominal distension. Leishmania amastigotes were found in the bone marrow smear. Again conventional treatment of pentavalent antimony was given for 6 days and his condition was improved. During hospitalization, the patient was screened for HIV antibody and found positive, which was further confirmed by Western blotting analysis. The CD4⁺ T lymphocytes was as low as 42 cells/ml. He was diagnosed with acquired immunodeficiency syndrome (AIDS). From July 2011, he began to receive conventional treatment including lamivudine (3TC, 300 mg/d) + zidovudine（AZT, 600 mg/d） + efavirenz (EFV, 600 mg/d). Two months later, AZT was replaced by tenofovir (TDF, 300 mg/d) due to severe anemia. On 25 July 2012, he was admitted to our hospital for “abdominal distension for two years”. Physical examination showed severe pallor and hepatosplenomegaly (the liver was 2 cm below the costal edge and the spleen was palpable 7 cm below the umbilical level). Leishmania was found in the bone marrow aspirate. Therefore, anti-HIV treatment with 3TC + TDF + EFV was continued, combined with sodium stibogluconate (pentavalent antimony 600 mg/day for 4 days, and 1 200 mg/day for an additional 4 days). After the liver and spleen began to shrink, the usage of stibogluconate therapy (pentavalent antimony 600 mg per time for 22 times) was changed every other day. Another two phase treatment with stibogluconate (pentavalent antimony 600 mg for 18 times) was given at one month and three months after the end of first phase treatment, respectively. The patient was given pentavalent antimony therapy with a total dose of up to 42 g during the three phase treatment, while also receiving conventional treatment on AIDS. Fifty months after pentavalent antimony withdrawal in April 2017, Leishmania was not found in the bone marrow, the CD4⁺ T lymphocytes was 225 cells/ml, and HIV-RNA was below detection limit. Conclusion Prolonged stibogluconate and conventional treatment can cure visceral leishmaniasis co-infected with HIV.

ORGINAL ARTICLES

CRISPR/Cas9-based localization and functional analysis of Toxoplasma gondii putative protein TGGT1_310420

Yong-gen JIA, Ai-xia YAN, Min-jun HUANG, Yang ZOU, Jun-chao GU

2019, 37(2):  150-155.  doi:10.12140/j.issn.1000-7423.2019.02.006

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Objective To characterize the function and localization of a putative protein TGGT1_310420 expressed on tachyzoite of Toxoplasma gondii. Methods The single guide RNA (sgRNA) targeting TGGT1_310420 was designed online. CRISPR/Cas9 construct targeting GGT1_310420 was obtained by mutating sgRNA of TgUPRT on pSAG1::CAS9-GFP-U6::sgUPRT. The knockout of TGGT1_310420 gene was performed through tagging the mCherry-Ty_HXGPRT sequence after the first 60 nt coding sequences of the endogenous copy using CRISPR/Cas9 genome-editing strategy. The correct insert was confirmed by PCR and DNA sequencing. The expressed fusion protein was detected by SDS-PAGE and Western blotting analysis, and its subcellular localization was determined by immunofluorescence microscopy. The phenotype of the knockout parasites was examined by a plaque assay. Results The CRISPR/Cas9 construct targeting TGGT1_310420 was successfully constructed and confirmed by sequencing. PCR analysis confirmed the integration of the mCherry-Ty_HXGPRT sequence into the correct locus and Western blotting assay detected a single protein band with an apparent relative molecular weight of 36 000. Immunofluorescent assay demonstrated that the first 20 amino acids of TGGT1_310420 fused to mCherry-Ty_HXGPRT was co-localized with T. gondii gliding associated protein 45(TgGAP45)to the parasite’s pellicle. Knockout of TGGT1_310420 gene revealed no measurable alteration in tachyzoites. Conclusion The first 20 amino acids of TGGT1_310420 are sufficient for pellicle targeting and the knockout of TGGT1_310420 did not change the phenotype of T. gomdii tachyzoite.

Protective immunity induced by recombinant surface protein-1 and surface protein-4 against Toxoplasma gondii infection in mice

Kai LIANG, Yan-wen LI, Bei-bei WANG, Xiao-yin FU, Xiao-quan LIU, Shan-shan HE, Li-li TANG, Deng-yu LIU, Huan-huan SHI

2019, 37(2):  155-160.  doi:10.12140/j.issn.1000-7423.2019.02.007

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Objective To determine the protective immunity induced by the immunization of recombinant surface antigen-1 (rSAG1) and surface antigen-2 (rSAG2) against Toxoplasma gondii infection in mice and compare their potential as vaccine. Methods The DNAs coding for T. gondii SAG1 and SAG4 were amplified by PCR from T. gondii genomic DNA and cloned into expression plasmid pET-28a(+). The success of cloning was confirmed by DNA sequencing, The recombinant plasmid DNAs were transformed into E. coli BL21(DE3) for expression as recombinant proteins under isopropyl-β-D-thiogalactopyranoside(IPTG) induction. The induced bacteria were lysed and the expressed rSAG1 and rSAG4 were purified by Ni-NTA resin under denatured condition. The denatured recombinant proteins were refolded by dialysis method. The refolded recombinant proteins were formulated with Freund’s adjuvant and used to immunize mice (n = 25 for each group) for 3 times. PBS and PBS + adjuvant were used as controls. Sera were collected after each immunization and specific IgG in the sera were measured by ELISA. Each mouse was challenged with 3 000 T. gondii tachyzoite two weeks after the final immunization. The survival time of the infected mice was observed and the data was analyzed using SPSS 16.0. Results The DNAs coding for full length SAG1 (780 bp) and SAG4 (438 bp) were successfully amplified from T. gondii genomic DNA and then cloned into pET-28a(+) to generate recombinant pET-28a(+)-SAG1 and pET-28a(+)-SAG4. The correct sequence and reading frame were confirmed by DNA sequencing. After being transformed into E. coli BL21 (DE3), both rSAG1 and rSAG4 were expressed as inclusion bodies. After being denatured with urea, the rSAG1 and rSAG4 were purified and refolded as soluble proteins. All mice produced high level of specific IgG in sera after being immunized three times with each recombinant protein compared to pre-immunization sera (P < 0.05). The ELISA A₄₅₀ value of rSAG1 group (1.821 ± 0.184) was significantly higher than rSAG4 group (0.695 ± 0.089) when sera were diluted at 1:400 (P < 0.05), but it is less than the combined group (rSAG1 + rSAG4) (1.955 ± 0.097) (P < 0.05). And the A₄₅₀ value of 3 experimental groups was higher than PBS (0.019 ± 0.002) group and PBS + adjuvant group (0.020 ± 0.004). All control mice (without immunization) died 224 h after being infected with 3 000 T. gondii tachyzoite, however, the mice immunized with rSAG1, rSAG4 and rSAG1 + rSAG4 survived till 296 h, 288 h and 320 h, respectively, after being challenged with the same number of T. gondii tachyzoite, with significant difference compared to the control group (P < 0.05). The increased survival time in the rSAG1 + rSAG4 group was significantly longer than each individual antigen group (P < 0.05). Conclusion Mice immunized with rSAG1, rSAG4 and rSAG1 + rSAG4 produced significant protective immunity against T. gondii infection with prolonged survival time than group without immunization. The co-immunization with both rSAG1 and rSAG4 produced synergic protective immunity compared to the individual antigen.

A modified method to infect Blastocystis hominis in rats and the pathological changes after infection

Jing LIU, Jyh-wei SHIN, Pei PEI, Xiao-yin FU, De-jun LIAO, Yun-yu LU, Sa XUE, Chen-chieh LIAO, Deng-yu LIU

2019, 37(2):  161-167.  doi:10.12140/j.issn.1000-7423.2019.02.008

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Objective To establish a rat model for Blastocystis hominis infection and lay a foundation for the further study of B. hominis. Methods Total 30 male Sprague-Dawley rats were randomly divided into four infection groups and one control group with 6 in each group. In the infection groups, the rats were infected with 10⁵, 10⁶, 10⁷ and 10⁸ of B. hominis trophozoites, respectively, by oral gavage. In the control group rats were given the same volume of PBS only. Three days after infection fecal samples were collected until 18 days after infection with once every three days. Total 5 fecal samples were collected for each rat. The fecal samples were cultured in LES medium for 72 hours and B. hominis in the culture were observed under microscope and confirmed by PCR. The genotype of B. hominis before and after infection was identified by DNA sequencing of small subunit rDNA (SSU rDNA). The number of infected rats was calculated and clinical sign was observed. On the 21st day after infection, all the rats were euthanized and the contents of the intestines including duodenum, jejunum, ileum, colon and cecum were collected and cultured in LES for 72 hours before microscopic examination of B. hominis in the culture. The final infection rate and infection intensity were calculated. The collected intestinal tissues were sectioned and stained by Hematoxylin-Eosin(HE) to observer the infection and pathological changes. Pearson correlation was used for the statistic analysis. Results Total 2, 3, 5 and 6 rates were confirmed to be infected with B. hominis in the groups infected with 10⁵, 10⁶, 10⁷ and 10⁸ B. hominis trophozoites, respectively. All nfected rats had been identified to be infected with B. hominis on the 3rd day after infection except for the rats in group infected with 10⁵ trophozoites that in which the parasites were identified on the 6th day post infection. No rats got infected in the control group. Sequence analysis of SSU rDNA amplified from the infected parasites confirmed that the subtype of the infected B. hominis was ST7. There was no mutation within the amplified SSU rNDA for the parasite before and after infection in the rats. In vitro culture of the intestinal contents showed that B. hominis parasites were found in the ileum, cecum and colon. The number of B. hominis identified in cecum and the cultured parasite in cecum was correlated with those identified in the colons(r = 0.541, P < 0.05). No parasite was found in other parts of intestine. HE staining of intestinal sections showed that B. hominis and mucin were found in the section of cecum, however, there was no invasion of parasite into mucosa, or lesion or erosion or inflammatory cell infiltration in the mucosa identified. Conclusion A rat model was successfully established for infection of B. hominis in our lab by oral gavage with as less as 10⁵ trophozoites, however it is more stable to establish infection in rats using 10⁸ of trophozoites. The more the trophozoites used for the infection, the higher infection rate can be obtained.

Polymorphism analysis of K13 gene of Plasmodium ovale isolates from Africa

Jing CHEN, Yao-bao LIU, Feng TANG, Feng LU, Jian-xia TANG, Jun CAO

2019, 37(2):  167-172.  doi:10.12140/j.issn.1000-7423.2019.02.009

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Objective To analyze the polymorphism of the K13 protein-encoding gene of Plasmodium ovale curtisi and wallikeri isolates from Africa so as to provide molecular basis for monitoring artemisinin resistance of P. ovale in Africa. Methods The blood samples were collected from imported malaria patients with P. ovale identified by microscopic and epidemiological investigation from 2012 to 2016 in Jiangsu Province. DNA was extracted from the blood samples and the P. ovale K13 gene was amplified by nested PCR. The subspecies of P. ovale were further determined by Real-time TaqMan^R PCR. The acquired DNA sequences were aligned with reference sequences from P. ovale curtisi subspecies (PlasmoDB: PocGH01_12019400) and the wallikeri subspecies (GenBank: LT594516.1) using BioEdit software. The mutations of the obtained DNA sequences were analyzed using DNAstar software. The polymorphism of K13 gene was analyzed by DnaSP software. The phylogenetic tree was constructed using MEGA software. Results A total of 168 P. ovale infected blood samples were collected from malaria patients during 2012-2016 in Jiangsu Province. It was confirmed that the sources of infection were from Central African (95 cases), South Africa (37 cases), West Africa (34 cases) and one case from East and North Africa. A 1 500 bp fragment of K13 gene was successfully amplified from all samples and sequenced. The sequence results identified that half of the samples were P. ovale curtisi subspecies (84) or wallikeri subspecies (84). The nucleotide diversity index π of P. ovale K13 gene was 0.000 02. The haplotype diversity index Hd was 0.024. The K13 genes of two subspecies contained two haplotypes and one single nucleotide polymorphism site. There is only one nucleotide polymorphism within each P. ovale subspecies. The P. ovale curtisi had a A/G polymorphism at nucleotide 717 (amino acid 239E) and the P. ovale wallikeri had a T/A polymorphism at the nucleotide 1998 (amino acid 666P), both of them were synonymous mutations without the change of encoded amino acids. The two mutations identified in this study are not related to P. ovale artemisinin resistance. Neutral test was performed on the DNA sequences of both subspecies of P. ovale without statistical difference. The mutation in P. ovale K13 gene in this study is in accordance with the neutral evolution model. The phylogenetic tree shows that the two haplotypes of P. ovale curtisi subspecies are clustered into one branch, and the two haplotypes of P. ovale wallikeri subspecies are clustered into another branch. Conclusion No nonsynonymous mutation is found in K13 gene of P. ovale curtisi and wallikeri subspecies in this study. K13 gene has a low level of genetic polymorphism in Africa isolations and no mutation is related to P. ovale artemisinin resistance.

Prevalence of Ascaris infection and the contamination of soil with Ascaris eggs in the rural areas of Ningxia during 2015-2016

Xiang-lin WU, Hong-ju DUAN, Yi-ren FU, Rong-ting QI, Jian-hua ZHAO

2019, 37(2):  173-177.  doi:10.12140/j.issn.1000-7423.2019.02.010

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Objective To investigate the prevalence of Ascaris lumbricoides infection and contamination of soil with Ascaris eggs in rural areas of Ningxia in order to provide evidence for developing control measures. Methods Total 52 survey sites in 14 counties (cities/districts) in Ningxia were randomly selected based on different geographic and economic situations during 2015-2016. At least 250 residents in each site were enrolled in the investigation. The fecal samples were collected from participants and the eggs of A. lumbricoides in the feces were examined in duplicate under microscope using Kato-Katz method and counted. To investigate the contamination of soil with A. lumbricoides eggs, 10 houses were randomly selected from each site and the soil was collected around their toilets and gardens, the eggs were examined using floating method with saturated sodium nitrate solution. Results A total of 13 850 persons were examined with overall A. lumbricoides infection rate of 2.2% (304/13 850). All infections were light without heavy infected people. People living in Loess Plateau Ecological Zone had higher infection rate of 3.1% （215/6 969） than those living in Longzhong Ecological Zone 1.3%（89/6 881） with statistical significance (P < 0.05). The top three counties with highest infection rates were Pengyang (9.3%, 116/1 250), Haiyuan (2.7%, 34/1 250) and Yanchi (2.6%, 20/760). Ascaris infection in Longde was moderate and the rest 13 counties were low-intensity infection （EPG < 500）. There was no significant difference in the infection rate between male (2.2%, 150/6 886) and female (2.2%, 154/6 964). People with age over 60 had higher infection rate of 3.2% than other age groups. The infection rate of people under 60 years old was increasing with age (P < 0.05). Han people had higher infection rate (2.6%, 214/8 373) than Hui (1.6%, 89/5 434), and infection in housewives (2.8%, 6/215) was higher than other occupations (P < 0.05). People with low education (elementary school) had higher infection rate (2.4%, 125/5 237) than those with higher education, but without statistical significance (P > 0.05). The soil samples were collected from garden and around toilet of 1 068 families and the Ascaris eggs were examined. The highest detection rate was 36.6% （37/101） in Pengyang. The lowest detection rate was 11.3% (9/80） in Yongning with statistical significance between different areas（P < 0.05）. Conclusion There was significant difference in the distribution of roundworm infection in different population such as age, nationality, occupation and education. The soil contamination with Ascaris eggs was common in Ningxia. It is needed to take different approaches to strengthen the control of Ascaris infection in different regions.

Endemic status of human common parasite infections in Qinghai Province in 2015

Jing-xiao ZHANG, Yu-fang LIU, Shi-lei CHENG, Rui DU, Na LIU, Wen LEI, Yong-shun WANG, Jia LIU, Pei-yun LIU, Hui-xia CAI, Xiao MA, Jun-ying MA, Wei WANG, Guo-ming SU

2019, 37(2):  178-182.  doi:10.12140/j.issn.1000-7423.2019.02.011

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Objective To understand the endemic status of human common parasite infections in Qinghai Province, so as to provide scientific basis for developing prevention and control measures. Methods The survey was performed in accordance with the National Survey Program and Implementation Rules to understand the status of human key parasite infections from May to September 2015. Total 51 survey sites were selected in 16 counties or cities based on a stratified cluster sampling method. Each site included no less than 250 permanent residents with age over 3 years old. The fecal samples were collected from each participant and the geohelminth eggs were examined under microscope using modified Kato-Katz method in triplicate. The intestinal protozoan trophozoites and cysts were examined using direct saline/iodine wet mount. The species of hookworm was identified by observing the larvae cultured from hookworm egg positive feces. The anal adhesive tape test was used to examine Enterobius vermicularis eggs for children with 3-6 years old. The collected data were statistically analyzed using SPSS 17.0 software. Results Totally 12 859 people were examined and overall prevalence of intestinal parasite infections was 2.0% (249/12 859), specifically 1.1%(138/12 859) for Ascaris lumbricoides, 0.9% (8/857) for E. vermicularis, and 0.8% (103/12 859) for intestinal protozoan infections (Entamoeba coli and Giardia lamblia), only one case was found with Taenia infection. The people living in Qilian mountain ecological region had the highest prevalence of soil-transmitted helminth (STH) (1.5%, 65/4 292) with statistical significance compared to that in other ecological regions (P < 0.01). For intestinal protozoan infection, people living in River source/Gannan ecological regions had the highest prevalence of 1.3% (56/4 302) with statistical significance compared to people living in other regions (P < 0.01). The infection rates of soil-transmitted nematodes for male and female people were 0.9% (59/6 435) and 1.2% (79/6 424), respectively. The infection rates of protozoan infections for male and female were 0.9% (56/6 435) and 0.7% （47/6 424）. There was no significant difference in the prevalence of intestinal parasite (STH or protozoan) between males and females (P > 0.05). People with age of 0-9 years old had the highest prevalence of STH (2.5%, 37/1 512) and people with 70-79 years old had the lowest STH infection rate (0.3%, 1/371), with statistical difference compared to other age groups (P < 0.01). People with age over 80 or between 10-19 had the highest prevalence of intestinal protozoan infections (1.3%, 1/78 and 1.2%, 20/1 673, respectively), however, the difference is not statistical significant compared to other age groups（P > 0.05）. For the prevalence in different nationality, Han and Tu had the highest prevalence for STH (2.1%, 99/4 707 and 1.0%, 5/486), and Mongol and Tibetan had the highest prevalence of intestinal protozoan infections (1.4%, 15/1 056 and 1.1%, 59/5 231), with statistical significance compared to people with other nationality (P < 0.01). The prevalence of STH was high in pre-school children (2.4%, 24/1 014), students (1.7%, 29/1 666) and farmers (1.4%, 72/5 336） with statistical significance compared with other occupations（P < 0.01）. The unemployed people had significantly higher infection rate of intestinal protozoan infections (4.8%, 1/21） than other people (P < 0.01). Conclusion A. lumbricoides infection is a major concern for the control of parasitic diseases in Qinghai, which are mostly distributed in agricultural-oriented areas and low-age populations. Intestinal protozoal infections are mostly distributed in animal husbandry areas with poor natural environment and poor living conditions.

Effectiveness evaluation of soil-transmitted nematode infection control in children in under-developed rural areas of Yi Nationality in Liangshan Prefecture, Sichuan Province based on the difference-in-differences estimation

Sha MENG, Chang SUN, Rui-xue YE, Yu-ju WU, Qing-zhi WANG, Min CAO, Yang-yang WU, Pu-chao LEI, Ling-ling LI, Huan ZHOU

2019, 37(2):  183-189.  doi:10.12140/j.issn.1000-7423.2019.02.012

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Objective The difference-in-differences (DID) estimation was used to accurately evaluate the effect of anti-parasitic disease intervention on children’s health-related behaviors and soil-transmitted nematode infection. Methods Children in Yi villages in Liangshan Yi Autonomous Prefecture, Sichuan Province, were selected based on a multistage stratified random cluster sampling method for the investigation in 2016. The demographic information of the participated children and their health-related behaviors were collected by a issued questionnaire. Fecal samples were collected from participated children with age ≥ 3 and the eggs of soil-transmitted nematodes were examined using Kato-Katz method under microscope. The children were randomly divided into the intervention and control group in the same village. A comprehensive intervention measures, including health education and anthelmintic treatment, were performed in the intervention group. One year after the intervention, the same method was used to collect the relevant information from the follow-upped children. Using the DID model, the accurate effect of the intervention on children’s health-related behaviors and the control of soil-transmitted nematode infection was analyzed. Results Total 162 children were recruited in this study with an average age of 7.7 years old. They were divided into the intervention group (99) and the control group (63). After the intervention, the DID analysis results demonstrated that the sanitation and hygiene behaviors have been significantly improved. These behaviors included the hand washing before meal at home (DID = 37.5%, P < 0.05), hand washing before meal in school (DID = 24.4%, P < 0.01), hand washing after toilet at home (DID = 45.3%, P < 0.01), hand washing after toilet in school (DID = 35.9%, P < 0.01) and brushing teeth and washing face (DID = 29.6%, P < 0.01). The positive effect was also reflected by the reduced infection rate of Ascaris lumbricoides (DID = -37.5%, P < 0.01). However, other DID values for some high-risk behaviors (eating unwashed fruits and vegetables, drinking un-boiled water, walking in barefoot, playing with mud on the ground and sucking fingers) and infection rates of hookworm and Trichuris trichiura were not significantly improved (P > 0.05). Conclusion After the interventions, some hygiene behaviors, as well as A. lumbricoides infection rate of school-age children has significantly improved. The DID estimation demonstrated the positive effect of interventions on the health-related behaviors and A. lumbricoides infection in children.

Endemic status of visceral leishmaniasis in China from 2004 to 2016

Shuai HAN, Wei-ping WU, Chui-zhao XUE, Wei DING, Yan-yan HOU, Yu FENG, Bo ZHONG, Lei CAO, Yu-nong ZHANG, Xiao-feng JIANG, Hong-wei ZHANG

2019, 37(2):  189-195.  doi:10.12140/j.issn.1000-7423.2019.02.013

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Objective To analyze the endemic status and epidemiological characteristics of visceral leishmaniasis in China from 2004 to 2016, so as to provide basis for the formulation of prevention and control strategies. Methods The data for the visceral leishmaniasis endemic in China from 2004 to 2016 were collected from Infectious Disease Reporting Management System of China Center for Disease Control and Prevention(CDC) and analyzed by Microsoft Excel 2016. For those cases reported in non-endemic areas, the infection information will be confirmed and verified by the local provincial CDC. Results A total of 4 448 cases of visceral leishmaniasis were reported in 24 provinces (municipalities or autonomous regions) in China from 2004 to 2016, with specific case number of 306, 311, 270, 355, 518, 523, 375, 300, 219, 158, 293, 510 and 310 cases for each year, respectively. The visceral leishmaniasis cases were reported throughout the year, but the peak was among October to November in Jiashi endemic county and among March to May for the rest of endemic areas. Total 98.1% (4 364/4 448) of the reported cases came from endemic areas, mainly from Xinjiang （including Xinjiang Production and Construction Corps）, Gansu and Sichuan provinces which account for 95.6% of the reported cases nationwide. Only 84 cases of visceral leishmaniasis were reported in 17 non-endemic regions, accounting for 1.9% of the reported cases. The visceral leishmaniasis endemic areas were located in 83 counties in 7 provinces or autonomous regions, among them 46 endemic counties located in Xinjiang Autonomous Region. The case reported counties are increased from 43 in 1990 to 83 in 2016, with reemerging areas in Linzhou, Wushi and Shache counties in Henan province. Total 12 visceral leishmaniasis cases were reported in non-endemic areas in 2016, all were confirmed to be imported cases. Among the total reported cases, 2 712 were male and 1 736 were female with a ratio of male to female to 1.6:1. The average age of reported cases was 12.3, mainly under 5 years old which accounts for 54.9% of total cases. Children and farmers were the most vulnerable population for visceral leishmaniasis which account for 72.6% of reported cases(3 228/4 448). Conclusion Visceral leishmaniasis endemic is sporadic in the middle western areas of China, with sporadic outbreaks and reemergence in some areas. The reported cases are decreasing except for those outbreak areas, but the endemic areas is growing.

Cloning and expression of the nucleoside diphosphate kinase gene of Giardia lamblia

Yi-xue YUAN, Guo-xia ZHENG, Dan-dan WANG, Hang ZHUAN, Meng DU, Yun-hua WANG

2019, 37(2):  190-193. 

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The nucleoside diphosphate kinase of Giardia lamblia(GlNDPK) (Chinese strain C2) coding sequence was amplified by PCR using the genomic DNA as the template. The PCR product was inserted into the pET28a plasmid and transformed into E. coli JM109. The positive clones were picked up for identification and sequencing. The pET28a-GlNDPK recombinant plasmid was transformed into E. coli BL21 (DE3), and expressed under IPTG induction. The proteins expressed were analyzed by SDS-PAGE. Inclusion bodies were dissolved with 8 mol/L urea and the supernatant was collected and purified by Ni²⁺ affinity chromatography. The recombinant protein was verified by Western blotting using anti-His6 tag antibody. The results showed amplification of the GlNDPK coding sequence of Giardia lamblia (Chinese strain C2) by PCR, resulting in a product of 1 200 bp. The recombinant plasmid pET28a-GlNDPK was verified by PCR, single enzyme digestion, and sequencing. As expected, SDS-PAGE showed that the recombinant GlNDPK protein was expressed as an inclusion body with a M_r of 40 000. Western blotting showed that the recombinant GlNDPK could be recognized by anti-His6 tag antibody. Therefore, the coding sequence of GlNDPK gene and its recombinant protein have been obtained.

ORGINAL ARTICLES

Polymorphism analysis of internal transcribed spacer 1 of Leishmania isolates from different endemic areas in China

Fang-zhou CHENG, Chun-hua GAO, Yue-tao YANG, Jun-yun WANG

2019, 37(2):  196-201.  doi:10.12140/j.issn.1000-7423.2019.02.014

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Objective The polymorphism of the internal transcribed spacer 1 (ITS-1) sequence of Leishmania isolates from different endemic areas in China was analyzed to understand the genetic variation and relationship among different Leishmania isolates. Methods Total 10 Chinese Leishmania isolates, including SC6 and Cy isolates from endemic areas of mountainous visceral leishmaniasis in Sichuan/Gansu Province (Leishmania donovani）; 801 and KS-2 isolates from endemic areas of anthroponotic visceral leishmaniasis in Kashi, Xinjiang (L. donovani）; JIASHI-1, JIASHI-5 and XJ771 isolates from endemic areas of desert-type visceral leishmaniasis in Jiashi, Xinjiang （L. infantum）; KXG-Xu, KXG-Liu and KXG-927 isolates from endemic areas of cutaneous leishmaniasis in Karamay, Xinjiang (L. infantum), were maintained in the laboratory culture. The genomic DNAs were extracted from the cultured promastigotes and the ITS-1 was PCR amplified using gene-specific primers. The PCR products of amplified ITS-1 were sequenced and the obtained sequences from different isolates were aligned with reference ITS-1 sequences of Leishmania deposited in GenBank using GENEDOC software. The phylogenetic tree was constructed based on their genetic variation and relationship. Results A single band of ITS-1 with size of about 310 bp was amplified from all 10 Chinese Leishmania isolates. The sequence analysis identified that ITS-1 amplified from anthroponotic L. donovani 801 and KS-2 isolates contained 316 bp with sequence identical to ITS-1 sequence from L. donovani reference strain MHOM/IN/80/DD8 without any mutation. The ITS-1 amplified from desert-type L. infantum JIASHI-1, JIASHI-5 and XJ771 isolates were 313 bp with 1.6% sequence variation. The ITS-1 amplified from cutaneous L. infantum KXG-Xu, KXG-Liu and KXG-927 isolates were 312 bp with 1.6% sequence variation. The ITS-1 amplified from mountainous L. donovani Cy and SC6 isolates were 318 bp with 0.6% sequence variation. Phylogenetic analysis of the ITS-1 sequences showed that the 10 Leishmania isolates were branched into three subgroups and two clusters according to the different species and endemic areas. The L. infantum isolates from Jiashi, Xinjiang (JIASHI-1, JIASHI-5 and XJ771) were closely related and clustered as subgroup A. The L. infantum isolates from Karamay, Xinjiang (KXG-Xu, KXG-Liu and KXG-927) are clustered as subgroup B. Subgroup A and B are closely related to L. infantum reference as cluster Ⅰ. The L. donovani isolates Cy, SC6, 801 and KS2 are closely related as subgroup C, fallen into cluster Ⅱof L. donovani. Conclusion ITS-1 sequences of Leishmania isolates from different leishmaniasis endemic areas in China showed different polymorphism related to different Leishmania species and endemic areas.

Effect of health education on the awareness and control of clonorchiasis in primary school students

Men-bao QIAN, Zhi-hua JIANG, Xiao-qin GAN, Jia-guang ZHAO, Wei LI, Wei-jie ZHENG, Guo-li LV, Ting-jun ZHU, Xiao-nong ZHOU

2019, 37(2):  202-206.  doi:10.12140/j.issn.1000-7423.2019.02.015

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Objective To evaluate the effect of health education on the awareness and control of clonorchiasis in primary school students in the endemic areas. Methods This study was implemented in Binyang County, Guangxi from 2017 to 2018. One primary school was selected as intervention group while another as non-intervention control group. A baseline questionnaire survey was firstly implemented in all students to understand their knowledge, practice and awareness related to clonorchiasis and the danger to eat raw fishes. Then, the comprehensive health education was carried out in the intervention group including the demonstration of an education cartoon and bulletin board on the transmission, prevention and control of clonorchiasis, distribution of educational brochure and conducting an drawing and essay competition related to the knowledge of the diseases. These educational activities did not perform in the control school. Six months later, a similar questionnaire survey were conducted to evaluate the effect of the education. Results Total 251 students in intervention group and 153 in control group participated in both baseline and evaluation surveys. In the intervention group, the awareness for the transmission route, early symptoms, complications, and carcinogenicity of clonorchiasis from 15.1% (38/251), 6.8% (17/251), 4.8% (12/251) and 4.8% (12/251) in the baseline survey significantly increased to 92.4% (232/251), 43.4% (109/251), 94.8% (238/251) and 57.8% (145/251), respectively, in the evaluation survey 6 months later, with statistical significance (P < 0.01). In contrast, in the non-intervention control group, the awareness for transmission route, early symptoms, complications, and carcinogenicity of clonorchiasis from 9.2% (14/153), 0.7% (1/153), 2.6% (4/153) and 2.6% (4/153) in the baseline survey increased to 56.9% (87/153), 33.3% (51/153), 42.5% (65/153) and 20.9% (32/153), respectively, in the evaluation survey, however, the increase was not as much as the intervention group. In intervention group, 16 students (16/251) who had history to eat raw freshwater fish during the past year in the baseline survey decreased to none to eat raw fish during the past 6 months. For those with no history to eat raw fish (235) in baseline survey, there was only one (1/235) who ate raw fish during the past 6 months. In the non-intervention control group, 35 students (35/153) who had history to eat raw freshwater fish during the past year in the baseline survey decreased to 15 to eat during the past 6 months. Out of 118 students who had not eaten raw fish, 6 had eaten raw fish during the past 6 months. The proportion of those who had history to eat raw fish but changed not to eat after baseline survey and those who had no history to eat raw fish but did after the baseline survey was significantly different between the two groups (P < 0.01). As to the awareness not to eat raw freshwater fish in future in intervention group, the rates for answering yes, no or not sure were 71.3% (179/251), 17.1% (43/251) and 11.6% (29/251), respectively, in baseline survey changed to 97.2% (244/251), 2.0% (5/251) and 0.8% (2/251) in evaluation survey (P < 0.01). In non-intervention control group, the rates of answering yes, no or not sure in the baseline survey [79.7% (122/153), 4.6% (7/153) and 15.7% (24/153)] had no significant change compared to the answers in evaluation survey [83.0% (127/153), 2.6% (4/153) and 14.4% (22/153)] (P > 0.05). Conclusion The comprehensive health education intervention in primary school students could effectively increase their knowledge and awareness to the transmission and prevention of clonorchiasis, as well as change their risk behavior to eat raw fish.

Analysis of clinical features on 15 case of fascioliasis

Wei-na ZHANG, Wei GU, Jian-ming JIAO, Jiao LUO

2019, 37(2):  207-212.  doi:10.12140/j.issn.1000-7423.2019.02.016

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Objective To analyze the general clinical features and manifestations, treatment and prognosis of fascioliasis. Methods The data of 15 patients diagnosed as fascioliasis and hospitalized at the First Affiliated Hospital of Dali University from July 2017 to March 2018 were collected. The general clinical features including manifestations, treatment and prognosis were analyzed. Results Total 15 patients were diagnosed as fascioliasis from July 2017 to March 2018. The patients came from different areas of Yunnan Province including 3 from Pu’er, 6 from Dali, 5 from Lincang and 1 from Lijiang. Four of them were males and 11 females. The first clinical symptoms were mainly abdominal pain (11 cases), fever (8 cases), abdominal bloating (5 cases), fatigue (3 cases), weight loss (4 cases), loss of appetite (2 cases), nausea (2 cases), vomiting (2 cases), etc.. The course of disease ranged from 1 d to 2 years with 8 cases happened in autumn, 4 cases in winter, 2 cases in summer, and 1 case in spring. Blood examination showed an increase in the percentage of eosinophils in 12 of 15 cases, 9 cases with decreased percentage of neutrophils, 7 cases with decreased lymphocyte count, 7 cases with increased platelet count and 6 cases with increased white blood cells count. In liver function tests, 10 cases showed an increase in alkaline phosphatase activity; 10 cases with increased γ-glutamyl transpeptidase. C-reactive protein was detected in 6 of 12 cases with the highest value of 154 mg/L. Most of the cases (11/12) had the elevated erythrocyte sedimentation rate with the highest of 120 mm/h. The eggs of Fasciola spp. were only found in the feces of 2 patients, however, all patients showed antibody positive in the ELISA test against Fasciola antigen. CT examination showed the diffuse multiple nodules and patchy lesions in the liver. Abdominal ultrasound showed liver heterogeneous echo mass. Histopathological examination of liver biopsy in 2 patients showed serious eosinophil infiltration, lymphocyte and eosinophilic abscess and local necrosis. Skin biopsy in one patient showed a large amount of eosinophil infiltration. Total 11 of 15 patients were misdiagnosed as liver abscess (2 cases), liver occupying lesions (2 cases), cholangio-carcinoma (2 cases), liver cancer (1 case), enteritis (1 case), connective tissue disease (1 case), acidophilic bacterial pneumonia (1 case), abdominal tuberculosis (1 case) in other hospitals. After being diagnosed as fascioliasis all 15 patients were treated with triclabendazole 10 mg/（kg·d) for successive two days. Symptoms improved significantly after treatment. The patients were followed up by telephone for 6 months, 10 patients with response showed good prognosis. Conclusion There still are cases of human infection with Fasciola spp. sporadically around Dali areas. More cases were seen in autumn and winter seasons. The clinical manifestations of fascioliasis are not specific and the infection is easily misdiagnosed. The immunological test with positive antibody is important for the diagnosis of fascioliasis. Triclabendazole is the first line of therapeutic drugs for fascioliasis.

Analysis of glutathione S-transferase gene expression in Pomacea canaliculata under Cu²⁺ stress

Guang-yao MAO, Hong-rang ZHOU, Yun HUANG, Zhi-yuan YUE, Lei DUAN, Qiu-li XU, Yun-hai GUO, Zhi-sheng DANG, Yi ZHANG, Ning XIAO

2019, 37(2):  213-218.  doi:10.12140/j.issn.1000-7423.2019.02.017

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Objective The expression of glutathione S-transferase gene in Pomacea canaliculata (PcGST) was analyzed under Cu²⁺ stress which is toxic to snail, and the related mechanism of P. canaliculata adaptation to Cu²⁺ stress was discussed. Methods Eighty snails were randomly divided into four groups. Four Cu²⁺ stress concentrations (0, 50, 100, 150 μg/L) were set to each group of 20 snails. Three snails from each group were randomly selected on 0, 1, 7, and 14 d after Cu²⁺ stress, and the liver, gill and kidney tissues were isolated. Total RNA were extracted from these tissues of P. canaliculata and reverse transcribed into cDNA. Real-time quantitative PCR was used to detect the relative expression of PcGST mRNA under Cu²⁺ stress. The PcGST gene was amplified from P. canaliculata tissue and then cloned into pET-28a expression plasmid. The recombinant plasmid pET-28a-PcGST was transformed into Escherichia coli BL21(DE3) bacteria and the recombinant PcGST was expressed under induction of isopropyl-β-D-thiogalactoside(IPTG). The expressed recombinant PcGST was analyzed by SDS-PAGE. The PcGST transformed E. coli BL21 and untransformed plain BL21 were equally divided into 6 aliquots. Three aliquots were randomly selected as Cu²⁺ stress group (with 0.2 mmol/L CuSO₄) and the other three as control group (without CuSO₄). All bacterial aliquots were cultured in a shaking incubator at 20 ℃ 150 r/min. A₆₀₀ of bacterial culture was measured every 1 hour until 6 hours. The tolerance of PcGST transformed and non-transformed bacteria to Cu²⁺ stress was compared by t-test. Results The relative expression of PcGST mRNA in P. canaliculata tissue without Cu²⁺ stress was set as 1.0 ± 0.0. In the liver tissue, under the stress of 50 μg/L Cu²⁺, the relative expression of PcGST mRNA increased continuously from 0 to 14 days, with a peak value of 4.9 ± 0.5, the relative expression of PcGST mRNA have gone up and then down, with a peak value of 13.0 ± 3.0 and 12.2 ± 2.2 on 7 d under 100 μg/L and 150 μg/L Cu²⁺ stress, respectively. In the gill tissue, the relative expression of PcGST mRNA have gone up and then down, with a peak value of 5.3 ± 0.7 and 23.3 ± 16.5 on 7 d under 50 μg/L and 100 μg/L Cu²⁺ stress, respectively, the relative expression of PcGST mRNA reached the peak of 9.8 ± 3.3 on 1 d then decreased under 150 μg/L Cu²⁺ stress. In the kidney tissue, the relative expression of PcGST mRNA did not increase at the early incubation under the stress of 50 and 100 μg/L Cu²⁺ till 7 d and reached the peak of 3.9 ± 1.0 under the stress of 100 μg/L Cu²⁺, the relative expression of PcGST mRNA reached the peak of 18.0 ± 0.8 on 14 d under the stress of 150 μg/L Cu²⁺. The The PcGST DNA was successfully amplified from P. canaliculate tissue with a size of approximate 600 bp and cloned into expression vector pET-28a. After being expressed in E. coli BL21, the expressed recombinant PcGST was appeared as 26 370 on SDS-PAGE. The PcGST transformed E. coli BL21 were grown better than the E. coli BL21 without PcGST transformed in LB liquid medium containing 0.2 mmol/L, with A₆₀₀ of 1.5 ± 0.1 and 1.0 ± 0.0, respectively, after 6 h culture, with statistical difference (P < 0.05), while the PcGST transformed and untransformed bacteria grew at the similar rate in LB liquid medium without Cu²⁺.Conclusion PcGST gene was upregulated in P. canaliculate tissue under Cu²⁺ stresss.

REVIEWS

Effects of soluble egg antigens on hepatic stellate cells in the progression of schistosomiasis-associated liver fibrosis

Xin LIU, Yong-fen QI, Yan-rong YU

2019, 37(2):  218-222.  doi:10.12140/j.issn.1000-7423.2019.02.018

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Schistosomiasis is a zoonotic parasitic disease worldwide caused by the infections of Schistosoma haematobium, S. mansoni and S. japonicum. The schistosomiasis endemic in China is only caused by S. japonicum infection that results in the deposition of eggs in the liver and causes the egg granuloma and eventually the liver fibrosis. The soluble egg antigens secreted by the miracidium induce host strong immune responses that contribute to the egg granuloma and liver fibrosis. Hepatic stellate cells are the central part of liver fibrosis. The regulation of soluble egg antigens on hepatic stellate cells plays important roles in the development of the fibrosis. This article reviews the research progress in the roles of soluble egg antigens on hepatic stellate cells in the progression of schistosomiasis-associated liver fibrosis.

Advances in ubiquitination modification of apicomplexa protozoans

Hao-rui LI, Hai-bin HUANG, Gui-lian YANG

2019, 37(2):  223-227.  doi:10.12140/j.issn.1000-7423.2019.02.019

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Apicomplexa phylum is a family of specific intracellular parasitic protozoans that undergo complex life cycles involving multiple developmental stages with distinct morphologies. Post-translational modification (PTM) occur throughout the complex life cycle of apicomplexan to regulate cellular processes and play roles in all aspects of cytobiology. Ubiquitination is a common modification in PTM. Protein ubiquitination plays a key role in protein turnover, cell signaling and intracellular transport. In this review, we summarize the ubiquitination modification of important organelles in apicomplexa protozoans to better understand the effect of ubiquitination on the cell cycle and functions of apicomplexan.

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Analysis of a case report of pulmonary cystic echinococcosis in a child in Jiangxi Province

Zhi-hong GONG, Yun XU, Ke-xing LIU, Kun-jiao DAI, Xiao-jun ZENG, An NING, Hui-qun XIE, Ai-ming QI, Cheng-jian HUANG, Yong-hong TU, Jing XU, Zu-lu GAO, Wei-sheng JIANG

2019, 37(2):  228-231.  doi:10.12140/j.issn.1000-7423.2019.02.020

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To better understand the clinical and epidemiological characteristics of pulmonary echinococcosis recently diagnosed in a child with 6 years old in Xinyu, Jiangxi Province, the related clinical manifestations and examinations (imaging, pathological, immunological results) and epidemiological data were collected and analyzed. Data analysis showed that the clinical diagnosis of the child was pulmonary cyst with parasitic infection. The serological test showed the antibody IgG positive to Cysticercus cellulosae antigen and Echinococcus antigen. The pathological biopsy identified Echinococcus granulosus parasite in the lung tissue. After removal of the pulmonary cyst by surgery, the child was treated with 0.2 g albendazole tablet twice a day for 3 consecutive months. The child was totally recovered with no relapse. The epidemiological investigation showed that the child had no history traveling to any endemic area of hydatid disease or exposing to any people or objects from endemic areas. Further investigation on 204 local residents living with the child identified no any sign of hydatid disease by abdominal ultrasonography. However, the serological test demonstrated that the positive rate of anti-Echinococcus antibody was 4.04% (8/198). The questionnaire survey showed that most of the children with low age usually had close contact with the dogs around. Fecal samples were collected from 9 dogs in the village and none of them had been detected with Echinococcus antigen. Based on the clinical and epidemiological data the child was diagnosed as a local infection of E. granulosus, suggesting the possibility of local transmission of local hydatid disease in this village.

Application of fluorescence quantitative PCR in laboratory diagnosis of malaria

Su-hua LI, Jing LI, Li-jun GAO, Ya-lan ZHANG, Rui-min ZHOU, Dan QIAN, Cheng-yun YANG, Ying LIU, Yu-ling ZHAO, Hong-wei ZHANG

2019, 37(2):  232-234.  doi:10.12140/j.issn.1000-7423.2019.02.021

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Seventy-nine malaria samples were examined by microscopy, nested PCR and fluorescence quantitative PCR, and the detection rate was analyzed among the three methods. After combining clinical symptoms of the patients and results of the three methods, 74 of the 79 samples were confirmed to be malaria infection, with a positive rate of 93.7%（74/79）. Specifically, the detection rate of microscopy, nested PCR and fluorescence quantitative PCR were 82.3%（65/79）, 82.3%65/79） and 93.7%（74/79）, respectively. The concordance rate between microscopy and nested PCR was 78.4%（58/74）, and that between microscopy and fluorescence quantitative PCR was 63.5%（47/74）, between nested PCR and fluorescence quantitative PCR was 83.8%（62/74）. In addition, the detection rate of fluorescence quantitative PCR was significantly higher than that of the nested PCR（P < 0.05）. Compared with microscopy and nested PCR, the fluorescence quantitative PCR has higher sensitivity and positive detection rate, and is less time-consuming.

Experimental observation of the development of Trichinella spiralis muscle larvae in mice

Guo-ying WANG, Xiang-hui LI

2019, 37(2):  235-237.  doi:10.12140/j.issn.1000-7423.2019.02.022

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To observe the developmental process and characteristics of Trichinella spiralis muscle larvae in mice, 28 female Kunming mice were orally infected with 100 T. spiralis muscle larvae and each two mice were euthanized at 8, 9, 10, 11, 13, 17, 20, 23, 27, 29, 50, 60, 240 and 400 days post infection. The diaphragm and partial abdominal muscles were collected, sectioned and stained with alum carmine acetate staining. The muscle larvae, larval capsules were observed under microscope. The muscle larvae were found in the diaphragm as early as 10 days post infection (10 d) and in abdominal muscles at 13 d, mostly with straight shape. The larvae were shaped in curve, circle, folded and larval capsules were formed at 17 d. The capsular bags were well developed, the stripes were seen in the wall of the capsule, and the capsule structure was divided into two layers without clear boundary at 29 d. The larvae twisted into a mass, and the structure inside the capsule began to appear transparent at 60 d. The capsule started to degenerate and the wall of the capsule began to appear transparent at 400 d. The average size of muscle larvae was (131.7 ± 21.4) μm × (10.4 ± 2.5) μm at 10 d, (435.5 ± 201.5) μm × (22.8 ± 5.2) μm at 17 d, (928.4 ± 188.5) μm × (35.0 ± 5.2) μm at 29 d and (1044.4 ± 86.7) μm × (38.3 ± 3.0) μm at 60 d, with significant difference in size between each developmental time points (P ﹤ 0.05 or P ﹤ 0.01). According to the experimental results, larval and capsular development can be roughly divided into four stages: pre-encapsulated larvae, larvae with capsular development, larvae with mature capsular bag, and larvae with degraded capsular bag. The morphological structure of larvae and capsules is closely related to the time of infection.

Effect of met-enkephalin on phagocytosis of Oncomelania hupensis hemocytes

Xiang-hong WEI, Ping TAN, Jin-hua WAN, Yan-bing SUN, Shi-wen LE, Qian-wen TIAN, Na LIU, Han-bing BAO, Yue SHEN

2019, 37(2):  238-241.  doi:10.12140/j.issn.1000-7423.2019.02.023

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To observe the effect of Met-enkephalin (MENK) on phagocytosis of hemocytes of Oncomelania hupensis, the hemolymph was obtained by squeezing the soft tissues of O. hupensis. The hemocytes in the hemolymph were incubated with different concentrations of MENK (0, 1, 5 and 10 mg/L) in the presence or absence of formaldehyde-treated sheep erythrocyte (FSE) at room temperature for 60 min. The hemocytes were stained with acridine orange and observed under fluorescence microscope. The results showed that the low concentration of MENK (1 mg/L) significantly stimulated the development of pseudopodia in the treated hemocytes of O. hupensis. The rates of pseudopodia development in the group treated with 1 mg/L of MENK was 48.77% (without FSE) and 48.3% (with FSE) with statistical significance compared to the control group (39.99%)(P < 0.01). The low concentration of MENK also stimulated the phagocytosis of treated hemocytes to FSE (48.77%) compared to control group without treatment (40.09%) with significant difference (P < 0.01). However, the high concentration of MENK (5 mg/L or 10 mg/L) not only inhibited hemocytes of Oncomelania to develop pseudopodia [27.04% (5 mg/L), 26.29% (5 mg/L + FSE) , 19.23% (10 mg/L) and 18.3%(10 mg/L + FSE) with significant difference compared to control group(χ² = 99.366, 109.74, 244.446, 274.34, respectively, all P < 0.01), but also dramatically inhibited the phagocytosis rate of treated hemocytes to FSE[7.37% (5 mg/L) and 5.8% (10 mg/L)] compared to 40.09% in untreated control group with statistical significance (P < 0.01). The results demonstrate that low concentrations of MENK can stimulate, but high concentration of MENK can inhibit the phagocytosis of treated hemocytes of O. hupensis.