Table of Content

28 February 2014, Volume 32 Issue 1

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In vivo Indentification of the Interaction Between var Intron and an ApiAP2 Transcription Factor in Plasmodium falciparum

HAN Shi-tong, ZHANG Qing-feng, PAN Wei-qing*

2014, 32(1):  1-01-05. 

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【Abstract】 Objective  To investigate the potential interaction between the ApiAP2 protein family member, PF3D7_1107800, and var intron of Plasmodium falciparum in vivo.  Methods  Genomic DNA was extracted from Plasmodium falciparum（3D7 strain）, 5′ end gene fragment of PF3D7_1107800 was amplified by PCR, and cloned into pGEX-4T-1 vector. The constructed pGEX-4T-1-PF3D7_1107800N was transformed into E. Coli BL21（DE3）　and followed by expression of the protein induced by IPTG. The recombinant protein was purified through glutathione sepharose. Twenty female BALB/c mice were divided into 2 group. Ten mice in experiment group were immunized with a mixture of the purified protein and Freund’s adjuvant by subcutaneous injection. Other 10 mice received PBS injection as control. Sera from mice of 2 group were purified with protein G. The effect of the antibody was testified with Western blotting. DNA products of ChIP assay was analyzed for enrichment of anti-PF3D7_1107800 group in ups C var intron by qPCR.  Results  PCR result of the PF3D7_1107800 gene 5′　end segment showed that there was a specific band（about 345 bp）, which was consistent with the theoretical value. The constructed vector pGEX-4T-1-PF3D7_1107800N was confirmed by gene sequencing. SDS-PAGE and Western blotting analysis demonstrated that the recombinant protein was about Mr 37　000. The anti-PF3D7_1107800 serum was obtained after the immunization of mice with the purified protein, and reacted with the recombinant protein, the specific band was about Mr 200 000. qPCR result showed that the fold enrichment of anti-PF3D7_1107800 group in var intron was two times higher than that of the reference gene region.  Conclusion 　The Plasmodium falciparum ApiAP2 family member PF3D7_1107800 binds to ups C var intron region in vivo.

Accumulation of Myeloid-derived Suppressor Cells in the Spleen and Peripheral Blood of Schistosoma japonicum-infected Mice

PAN Wei，SHEN Yu-juan，LIU Hua，HU Yuan，JIANG Yan-yan，ZHOU He-jun，XU Yu-xin，

2014, 32(1):  2-06-11. 

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

Effect of Schistosoma japonicum Infection on Serum Lipid Status in Mice

XUE Yan-feng，SHEN Li*

2014, 32(1):  3-12-17. 

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【Abstract】 Objective  To investigate the effect of Schistosoma japonicum infection on lipid status in mouse serum.  Methods  Twenty-four ICR mice were randomly divided into two groups, fed a high fat diet（HFD） or a normal diet（ND）. On the 28th day, 6 mice from each group were infected with double sex cercariae of S. japonicum via abdominal skin（150 cercariae/mouse）. At 42 days post-infection, the mice were sacrificed and the sera were collected. Other 36 ICR mice were randomly divided into three groups fed on normal diet. Mice in the first group were infected with S. japonicum single-sex cercariae via abdominal skin（150 cercariae/mouse） and sacrificed on Day 42. Mice in the second group were intraperitoneally injected with 10 000 S. japonicum eggs and serum samples were collected at Day 4 and Day 7. Mice in the third group were intraperitoneally injected with soluble egg antigen（SEA） every day for 6 days［1 mg/（mice·d）］ and serum was collected at Day 7. Mice from control group were fed a high fat diet or a normal diet without infection. Serum levels of total cholesterol（TC）, triglyceride（TG）, high-density lipoprotein（HDL）, and low density lipoprotein （LDL） were measured.  Results  Compared with the uninfected controls, the serum levels of TC, TG, HDL, and LDL decreased significantly in double-sex cercariae infected-mice fed on a high fat diet or a normal diet （P＜0.05）. In HFD group, serum TC[（1.45±0.31)mmol/L], TG[（0.17±0.06)mmol/L], HDL[（1.11±0.26)mmol/L] and LDL[（0.44±0.15)mmol/L] levels in mice infected with double-sex cercariae were lower than that of uninfected mice[（7.86±0.07)mmol/L, (0.23±0.07)mmol/L, (4.96±0.81)mmol/L, (3.93±0.29)mmol/L]（P＜0.05）. In ND group, serum TC[（1.03±0.08)mmol/L], TG[（0.17±0.03) mmol/L], HDL[（0.84±0.02)mmol/L], and LDL[（0.09±0.02)mmol/L] levels in mice infected with double-sex cercariae were lower than that of uninfected mice[（1.85±0.05)mmol/L, (0.90±0.14)mmol/L, (1.38±0.18)mmol/L, (0.15±0.01)mmol/L, respectively]（P＜0.05）. The mice serum lipid indices had no obvious change after single-sex cercariae or egg injection（P＞0.05）. Serum TC[（1.07±0.15) mmol/L], TG[（1.06±0.15)mmol/L], HDL[（0.71±0.14)mmol/L], and LDL[（0.05±0.04)mmol/L] levels in SEA injected mice were lower than that of the control group[（1.81±0.06)mmol/L, (2.15±0.13)mmol/L, (1.16±0.15)mmol/L, (0.16±0.03)mmol/L]（P＜0.05）.  Conclusion  Schistosoma japonicum infection can decrease serum lipid concentrations in the mouse host.

Effect of RNA Interference Targeting Schistosoma japonicum Aldose Reductase Gene

WANG Shu-qi1，XU Bin2，LIU Jian1，WANG Ji-peng1，HU Wei1，2 *

2014, 32(1):  4-17-21. 

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【Abstract】 Objective  To investigate the effect of siRNA-induced RNA interference（RNAi） at both messenger RNA（mRNA） and protein level, targeting the Schistosoma japonicum aldose reductase （SjAR） gene.  Methods  Two siRNAs （siRNA-1 and siRNA-2） were designed based on SjAR gene sequence and commercially synthesized together with a negative control siRNA（siRNA-NC） showing no homology to any known S. japonicum gene sequences. Each female Kunming mice（4-6 weeks old） was infected with 800-1　000 S. japonicum cercariae and sacrificed on Day 13 post-infection for schistosomula collection. Every 120±10 worms were selected as one group and subjected to RNAi treatment based on siRNA electroporation or soaking. In terms of the electroporation method, 5 μg of siRNA-1, siRNA-2, siRNA-NC or equal volumes of DEPC water （blank control） were added to each cuvette separately and the schistosomula in the cuvettes subsequently underwent a square wave pulse of 125 V and 20 ms duration, followed by a 48 h culture. As for the soaking method, solutions of the three siRNAs were added to the culture media separately to a concentration of 200 nmol/L, with equal volumes of DEPC water added as a blank control. Fresh media were changed and siRNAs were supplemented on Day 3 amid the 5 days′ soaking process. Three repeats were set up for each treatment. Worm RNA and total soluble proteins were extracted after the RNAi treatment by using the TRIzol method, and cDNA was synthesized by reverse transcription of the RNA. Quantitative real-time PCR（qRT-PCR） was performed next to determine the change of relative SjAR transcripts abundance after the RNAi treatment with SjGAPDH as an internal reference. Western blotting was performed to measure the expression changes of SjAR at protein level, after degradation of the SjAR transcripts by RNAi treatment.  Results  qRT-PCR result showed that the relative expression level of SjAR transctripts was (90.6±16.2)%, (84.2±7.3)%, and (105.9±10.3)%, respectively, following the electroporation of siRNA-1, siRNA-2 or siRNA-NC as well as the 48 h culture, none of which showed a statistically significant difference compared with the blank control group [（100.9±16.8)%]（P>0.05）. However, after schistosomula soaked with siRNA-1 and siRNA-2 for 5 days, the levels of SjAR transcripts were reduced to (48.6±8.2)% and (73.4±4.7)%, respectively, both showing a statistically significant difference in comparison to the blank control group[（100.04±3.25)%]（P<0.01）. The siRNA-NC treated group （negative control） exhibited no statistically significant difference compared with the blank control group（P>0.05）. Accordingly, as shown by Western blotting, the SjAR protein levels of worms soaked with siRNA-1, siRNA-2 or siRNA-NC were 79.0%, 97.8% and 103.2%, respectively, compared with the blank control.  Conclusion  RNAi treatment based on siRNA soaking reduce the expression of SjAR gene at both mRNA and protein level.

Gene-cloning, Expression and Immunoreactivity Detection of Toxoplasma gondii Uridine Phosphorylase

YIN Li-tian1，ZHU Jian-jiang2，LI Run-hua3，WANG Hai-long2，LI Ya-qing2，YIN Guo-rong2 *

2014, 32(1):  5-24-28. 

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 【Abstract】 Objective  To predict the physicochemical properties and antigenic epitopes of Toxoplasma gondii uridine phosphorylase（TgUPase）, clone, and express TgUPase gene, and analyze its immunoreactivity.  Methods  The physical and chemical characters and specific epitopes of TgUPase protein were predicted by bioinformatics software tools. Total RNA was extracted from RH strain T. gondii tachyzoites. A pair of specific primers was designed according to the open reading frame of TgUPase gene （GenBank Accession No. DQ385446.1）. RT-PCR product was digested with restric-tion enzyme and ligated into a pET-30a（+） vector. The recombinant plasmid pET-30a（+）-TgUPase was transformed into E. coli DH5α and the positive clones were selected by colony PCR and confirmed by double restriction enzyme digestion and sequencing. The constructed pET-30a（+）-TgUPase was then transformed into E. coli BL21（DE3） and induced with IPTG for expression. The expression product was analyzed through SDS-PAGE followed by Coomassie blue staining. Western blotting assay with His primary antibody and human anti-T.　gondii serum was used to confirm the expression of rTgUPase and detect its immunoreactivity.  Results  Bioinformatics prediction results showed that rTgUPase protein was 303 amino acids in length with a predicted molecular mass of Mr 33 042.9, and this soluble protein had three potential T/B cell epitopes. The product of RT-PCR was 921 bp. Colony PCR, double restriction enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-30a（+）-TgUPase was constructed. SDS-PAGE showed that bacteria containing recombinant plasmid pET-30a（+）-TgUPase expressed a soluble protein of His-TgUPase （about Mr 38 000） after being induced with IPTG. The recombinant protein reacted positively with His primary antibody and human anti-T.　gondii serum by Western blotting analysis.  Conclusion  The recombinant plasmid pET-30a（+）-TgUPase is constructed and the soluble rTgUPase shows immunoreactivity.

Construction，Expression and Kinase Function Analysis of an Eukaryocyte Vector of Rhoptry Protein 17 in Toxoplasma gondii

WANG Hai-long1，YIN Li-tian2，ZHANG Tie-e1，GUAN Li1，3，MENG Xiao-li1，LIU Hong-li1，YIN Guo-rong1 *

2014, 32(1):  6-29-33. 

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【Abstract】 Objective  To construct and express the eukaryocytic expression vector of rhoptry protein 17 of Toxoplasma gondii RH strain(TgROP17) and analyze its kinase function.  Methods  The open reading frame of TgROP17 gene was amplified from total RNA in T. gondii RH strain by RT-PCR, and cloned into p3×Flag-CMV-14 vector to construct recombinant plasmid p3×Flag-CMV-14/TgROP17. After colony-PCR confirming, double restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector p3×Flag-CMV-14/TgROP17 was transfected into HEK 293T cells. The target gene was examined by RT-PCR and the recombinant protein was detected by Western blotting. The kinase activity of TgROP17 was identified by Western blotting and its apoptotic function was assessed by flow cytometry.  Results  The size of RT-PCR product was 1 850 bp. The recombinant plasmid p3×Flag-CMV-14/TgROP17 was confirmed by colony-PCR, double restriction enzyme digestion and DNA sequencing. RT-PCR and Western blotting analysis showed that TgROP17 was expressed in the p3×Flag-CMV-14/TgROP17 transfected-HEK 293T cells rather than in mock cells. The amplified gene was with 1 850 bp and the target protein was about Mr 70 000. Western blotting analysis showed that c-Jun was phosphorylated by TgROP17. Flow cytometry analysis indicated that camptothecin-induced apoptosis was inhibited by TgROP17 with an inhibition rate of 20.6% and 24.1% at 6 h and 12 h after co-culture, respectively, which was higher than that of the control(P＜0.05).  Conclusion  The eukaryotic expression vector p3×Flag-CMV-14/TgROP17 is constructed. TgROP17 has kinase activity and playes an anti-apoptosis role.

Preparation and Purification of Polyclonal Antibody against Toxoplasma gondii Rhoptry Protein 2（ROP2） and its Application in Immunofluorescence Localization

ZHANG Li，LIAO Qi-Bin，HU Lin，CHEN Ai-Yuan，WEI Hai-Xia，PENG Hong-Juan*

2014, 32(1):  7-34-37. 

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 【Abstract】 Objective  To prepare and purify polyclonal antibody against Toxoplasma gondii rhoptry protein 2 (ROP2) and apply it to immunofluorescence localization.  Methods  The constructed recombinant plasmid pET32a-ROP2 was transformed into E. coli BL21（DE3） and the protein was expressed under the condition of 0.5 mmol/L IPTG induction. Cells were lysed by multiple rounds of sonication to obtain supernatant and inclusion body, respectively. The washed inclusion bodies were dissolved in urea. New Zealand White rabbits were immunized with 200 μg purified recombinant ROP2 mixing with the same volume of Freund′s adjuvant for 3 times at interval of 14 days and 19 days, respectively. Rabbit serum was collected at 10 days after the last immunization. Polyclonal antibody in rabbit serum was purified with HiTrap Protein G HP affinity purification column. Indirect ELISA and Western blotting were used to detect antibody titer and specificity of polyclonal antibody against the recombinant ROP2. The polyclonal antibody was used to the localization of ROP2 on the parasitophorous vacuole membrane in human foreskin fibroblasts infected by Toxoplasma tachyzoites by the immunofluorescence method.  Results  The recombinant ROP2 protein was obtained and specific rabbit-derived polyclonal antibody was prepared. Indirect ELISA confirmed that the rabbit-derived polyclonal antibody titer reached 1 ∶ 102 400, and the recombinant ROP2 protein was recognized by specific polyclonal antibody. Immunofluorescence localization test showed that the ROP2 protein was located on the parasitophorous vacuole membrane.  Conclusion  The rabbit-derived polyclonal antibody against ROP2 is prepared, and used in immunofluorescence localization of ROP2 on parasitophorous vacuole membrane.

Cloning and Expression of Transferrin Protein from Culex pipiens pallens and a Study of its Antimicrobial Activity

WU Song-quan1 *，WANG Guang-li1，ZHOU Wu1，ZHONG Shi-gen2

2014, 32(1):  8-38-41. 

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 【Abstract】 Objective  To clone and express transferrin（Tsf） from Culex pipiens pallens in Pichia pastoris，and detect its antibacterial activity.  Methods  The coding region of transferrin from Culex pipiens pallens was amplified by RT-PCR. The product of RT-PCR was inserted into the downstream of gene encoding α-factor signal sequence in a Pichia pastoris secreting expression vector pGAPZα-A. The recombinant pGAPZα-A-Tsf vector was transformed into P. pastoris GS115 by electroporation. Recombinant strains pGAPZα-A-Tsf/GS115 were screened by Zeocin resistance and PCR. Recombinant protein was detected by SDS-PAGE and Western blotting. The recombinant transferrin protein was purified by using Ni-NTA resin. The antibacterial activity of the purified transferrin against Escherichia coli was detected.  Results  The transferrin gene with 2 100 bp was obtained by RT-PCR. The product of recombinant plasmid pGAPZα-A-Tsf was approximately 2 127 bp by double digestion with restriction enzymes, consistent with the anticipated fragment length. Sequencing results showed that the inserted sequence was correct. PCR result showed that the recombinant plasmid pGAPZα-A-Tsf/GS115 was constructed. The results of SDS-PAGE and Western blotting showed that the relative molecular weight (Mr) of the protein was about 80 200. The recombinant transferrin protein showed antibacterial activity against Escherichia coli, and the minimum concentration was 0.25 mg/ml.  Conclusion  The recombinant transferrin protein from Culex pipiens pallens has been expressed in P. pastoris, and shows antibacterial activity against E. coli.

Study on Ficolin-A against Infection of Plasmodium berghei in Mouse Model

XIANG Tian1, LIU Gao1, DAI Wan-an1, LI Zong-qing1, CHEN Fan2 *

2014, 32(1):  9-42-45. 

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 【Abstract】 Objective  To evaluate the effect of Ficolin-A, a lectin complement against Plasmodium berghei in mice model.  Methods  The Mr 19 000 fragment of merozoite surface protein-1 of P. berghei（MSP119） was cloned and then subcloned into the vector pGEX-KG. The recombinants of pGEX-KG-Ficolin-A and pGEX-KG-MSP119 were transformed into Escherichia coli BL21, and followed by expression of the protein induced by 1 mmol/L IPTG. The fusion protein was purified by affinity chromatography using Glutathione Sepharose 4B, and then identified by SDS-PAGE and Western-blotting. Five mouse model groups were treated with PBS, GST, Ficolin-A, MSP119, or Ficolin-A+MSP119, respectively. Each group had eight mice. Mice in Ficolin-A or MSP119 groups were injected with 20 μg Ficolin-A or MSP119 protein each time, respectively. Mice in Ficolin-A+MSP119 group were injected with 20 μg Ficolin-A and 20 μg MSP119 each time. Mice in control groups were injected with 200 μl PBS or 20 μg GST, respectively. All the mice received four immunizations at 2-week intervals. Two weeks after the last immunization, all the mice were inoculated with 300 μl Plasmodium berghei-infected red blood cells. On day 2, 4, 6, 8, and 10 post-infection, blood samples were collected from three mice of each group, and the Giemsa stained-blood films were microscopically examined. Density of malaria parasites was calculated. The survival rate was evaluated on day 20 post-infection.  Results  The recombinant vectors of pGEX-KG-Ficolin-A and pGEX-KG-MSP119 were constructed. Purified fusion proteins, Ficolin-A-GST and MSP119-GST, were obtained. Western blotting analysis indicated that the relative molecular mass of fusion proteins Ficolin-A-GST and MSP119-GST was about Mr 69 000 and Mr 41 000. Animal experiments showed that on day 10 after infection, the parasite density in Ficolin-A+MSP119 group ［（22.2±1.7）%］ was slightly lower than that of the groups MSP119 ［（33.4±2.7）%］, Ficolin-A [（36.2±3.1）%］, GST ［（43.8±4.8）%］ and PBS [（45.3±3.6）%], but the difference was not statistically significant（P>0.05）. No mouse survived in PBS group on day 20 after infection. There was no significant difference in number of survival mice between Ficolin-A group (3 mice) and GST group  (2 mice). Six mice survived in Ficolin-A+MSP119 group, which was significantly more than that of GST group （P<0.05）.  Conclusion  Ficolin-A cannot significantly suppress parasite density. However， Ficolin-A+MSP119 can increase the survival rate of Plasmodium berghei-infected mice.

Assessment of Malaria Knowledge and Capability on Plasmodium Detection among Professionals from Class Ⅲ Malaria-endemic Counties of Henan Province in 2012

ZHOU Rui-min，LIU Ying，Qian Dan，SU Yun-pu，HE Li-jun，YAN Qiu-ye, LU De-ling，YANG Cheng-yun，ZHAO Yu-ling，XU Bian-li，ZHANG Hong-wei*

2014, 32(1):  10-46-49. 

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【Abstract】 Objective  To evaluate the capability of malaria parasite detection among professionals from Class Ⅲ malaria endemic counties of Henan Province in 2012. 　Metheds  The capacity assessment of professionals from the Centres for Disease Control and the medical institutions was done in September to December 2012. The content of the assessment included  malaria knowledge （including malaria etiology, clinical manifestation, diagnosis, treatment and epidemiology and so on, 100 scores as full marks and 60 as passing score）, making blood slides of Plasmodium（4 slides in 1 hour, including Giemsa staining, 40 scores as full marks and 24 as passing score） and identification of species with microscopy （6 slides, 8 min per slide 60 scores as full marks and 36 as passing score）. All the participanats were grouped by gender, age, professional title, level and type of institution. Their scores were statistically analyzed by SSPS 17.0 software.　 Results  The average total score in 891 participants was 162.1, the highest was 200 (full markers), and the lowest was 96, and 871 （97.8%） participants passed the test (≥120 scores). There were no significant differences for the scores of blood slide making among gender, age, professional title and level of institution（P＞0.05）. No significant differences in the scores of malaria knowledge and blood slide reading among gender （P＞0.05）, however, the total score in female participants（162.97±17.64） was higher than that of males（159.01±20.33）（P＜0.05）. The film-reading and total scores of 50-plus age group （34.62±14.82, 144.62±20.33） was significantly lower than the other three groups （under age 30 group: 45.75±13.58 and 162.50±18.90, age 31～40 group: 46.53±12.72 and 163.51±17.77, age 41～50 group: 46.22±13.38 and 159.80±17.32）（P＜0.05）. The scores of malaria knowledge in 50-plus age group （84.38±9.41） was lower than that of under age 30 group （89.91±7.81）, age 31～40 group （89.96±7.74）（P＜0.05）. The scores of malaria knowledge （88.33±8.23, 90.00±7.76, 92.37±7.29）, film-reading （44.88±13.62, 46.59±12.88, 49.57±11.98） and total scores （159.61±18.37, 163.81±18.03, 169.15±16.38） of primary, intermediate and senior groups was proportional to the level of the titles, and the difference between the groups was statistically significant （P＜0.05）. The theory （88.28±8.30, 90.84±7.32, 93.54±6.10）, film-reading （44.54±13.14, 47.69±13.40, 52.62±11.04） and total scores （159.48±18.33, 165.92±17.31, 171.97±15.53） of the three institution level groups （township, county and province） were proportional to their level, and the difference between the groups was statistically significant （P＜0.05）. There was no significant differences for the scores between the CDCs and hospitals （P＜0.05）.  Conclusion  The capabilities of malaria parasite detection in Class Ⅲ malaria endemic counties of Henan Province is balanced. It needs to strengthen the skills training for the professionals of the junior, intermediate and primary care units.

Comparison of Two Rapid Diagnostic Tests in Detection of Malaria Parasites

WANG Zhen-yu，JIANG Li，ZHANG Yao-guang，ZHANG Xiao-ping，CAI Li*

2014, 32(1):  11-50-53. 

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【Abstract】 Objective  To compare the performance of two rapid diagnostic tests（RDTs） for malaria parasite detection.  Methods  Blood samples of 200 malaria patients and 60 non-malaria persons were collected from Yunnan and Shanghai, respectively. The sera were detected by gold-colloidal immunochromatography(GICA)and OptiMAL, and microscopy was used as gold standard in species identification. The sensitivity, specificity, minimum detection limit of the two RDTs was compared.  Results  Of the 260 samples, malaria parasites were found in 200 by microscopy, of which 100 each were Plasmodium falciparum and P. vivax, respectively. Compared with microscopy, the sensitivity and specificity of GICA and OptiMAL for the samples were 87.5%（175/200）and 93.3%（56/60）, 95.5%（191/200）and 100.0%（60/60）, respectively. The sensitivity and specificity of GICA and OptiMAL for detection of P. falciparum were 83.0%（83/100）and 96.9%（155/160）, 90.0%（90/100）and 99.4%（159/160）, respectively; and for detection of P. vivax, they were 89.0%（89/100）and 98.8%（158/160）, 96.0%（96/100）and 97.5%（156/160）, respectively. There was a significant difference in malaria detection between GICA and OptiMAL（χ2=8.23, P<0.05）. No statistical difference was found between the two RDTs in P. falciparum and P. vivax detection（P>0.05）. OptiMAL showed better result in detection of P. falciparum when the parasite density was higher. The minimum detection limit of the two RDTs was about 100-200 parasites/μl blood.  Conclusion  Compared to GICA, OptiMAL has higher sensitivity and specificity. However, GICA shows lower minimum detection limit and better reproducibility in blood samples with different densities than that of OptiMAL.

Tracing Investigation and Diagnosis of One Transfusion-transmitted Malaria Case in Zhejiang Province

RUAN Wei1，LEI Yong-liang2 *，YAO Li-nong1，ZHANG Ling-ling1，LIU Xiao-hong2，XIANG Xiao-qing3，MEI Jian-hua2，ZHU Hai-bo3，YU Yang2，ZENG Chang-you2

2014, 32(1):  12-54-57. 

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【Abstract】 Objective  To identify the sources of infection and the mode of transmission of a malaria case with unknown origin.  Methods  Clinical data of the case were collected and the epidemiological investigation was conducted. The blood samples of the patient and the suspected infection source(blood donor) were detected by microscopy, rapid diagnostic test strip(RDT)and nested PCR.  Results The patient did not visited malaria endemic areas. After a blood transfusion, the patient had chills and fever, and was confirmed as falciparum malaria by microscopy with bone marrow and peripheral blood smears and RDT. The blood donor was a worker returned from Africa. Before blood donation she was sick like malaria carrier, and took anti-malarial drug. She was then confirmed as falciparum malaria by RDT and microscopy. The blood samples from the patient and the blood donor were diagnosed as falciparum malaria by nested PCR, and the similarity of the small subunit rRNA(SSU rRNA) sequence was 100%, showing they were mix-infected with K1 and MAD20 genotypes of Plasmodium falciparum.  Conclusion  This patient is confirmed P. falciparum infection via blood transfusion from a donor who returned from Africa.

Pathological Changes of after Trans-Portal Vein Chemoembolization Echinococcus multilocularis in the Liver of Infected Rats

ZHU Di-wen1，ZHANG Hai-xiao1，REN Wei-xin1 *，XIONG Jin2，XU Xiao-hui2，WEN Hao3

2014, 32(1):  13-58-61. 

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【Abstract】 Objective  To observe the morphological change in the pathological process of Echinococcus multilocularis in liver of infected rats after treatment with liposome entrapped albendazole and iodized oil suspension through portal vein, and investigate its efficacy against hepatic alveolar echinococcosis.   Methods  One Echinococcus multilocularis- infected rat served as control. Another 19 infected rats were infused with 0.2 ml liposome entrapped albendazole and iodized oil suspension through portal vein. At 4, 7 and 10 d after treatment, the rats were sacrificed for further observation. The E. multilocularis metacestodes were collected, and the pathological change of alveolar hydatid was observed with hematoxylin-eosin staining and toluidine blue staining.  Results  In four days post treatment, E. multilocularis metacestodes were normal. On the 7th day and 10th day post treatment, the major pathological change was degeneration and necrosis, respectively. A great quantity of liposomes and iodized oil deposited in the metacestodes, and caused breakdown of E. multilocularis tissue, collapse of cysts, cataplasia of germinal layer and laminated layer.  Conclusion  Interventional therapy trans-hepatic vein may be an effective way to treat hepatic alveolar echinococcosis.

Overviews on Plasmodium ovale wallikeri and its Genetic Detection System

LI Mei，XIA Zhi-gui *，TANG Lin-hua

2014, 32(1):  14-64-67. 

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【Abstract】 In recent years, some microscopy-positive but nest-PCR negative（based on small subunit ribosomal RNA gene） ovale malaria cases were reported. The detection failure was due to the genetic variations between Plasomdoium ovale wallikeri and P. ovale curtisi. This paper reviews the discovery and validation processes of P. ovale wallikeri and the detection systems for identification of P. ovale.

Hemozoin Detection in Malaria Diagnosis

YAO Mei-xue, WANG Heng*

2014, 32(1):  15-68-71. 

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【Abstract】 In malaria parasites, hemozion is often called malaria pigment. Plasmodium spp. digest hemoglobin and release high quantity of free heme, which is a non-protein component of hemoglobin. Until recent years, researchers have found that hemozoin has many effects on malaria parasites. This paper reviews the hemozoin detection apllied in malaria diagnosis.

Research Progress on the Molluscicidal Effect of Niclosamide Compounded with Other Molluscicides against Oncomelania hupensis

SUN Feng，ZHANG Jian-feng，WEN Li-yong*

2014, 32(1):  16-72-75. 

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【Abstract】 Niclosamide is the only molluscicide recommended by the World Health Organization. It is effective for snail control but have some disadvantages. Therefore, the research goal is to increase the molluscicidal activity of niclosamide and  reducing its defects. This article reviews the research advances on the molluscicidal effect of niclosamide compounded with other molluscicides against Oncomelania hupensis.

Seasonal Changes of Glycogen Level in Oncomelania hupensis

HUANG Chun-lan1，ZHOU Shu-lin1 *，LI Chao-pin1，ZHAO Jin-song1，YANG Jin-sun2

2014, 32(1):  17-21-23、28. 

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【Abstract】 Oncomelania hupensis snails were collected from Qingyi River of Wuhu City from August 2012 to July 2013. Livers and pedal muscles of snails were dissected. Anthrone colorimetric method was used to evaluate the glycogen concentrations of whole-body, liver and muscle. The concentration of whole-body and liver glycogen decreased from September to next June. The whole body glycogen content in female （0.55 μg/mg） and male （0.88 μg/mg） snails was the lowest in June and April, respectively. The mean whole-body glycogen concentration in females and males was 2.99 and 3.39 μg/mg, respectively. Liver glycogen concentration was lowest in May（♀＝0.29 μg/mg，♂＝0.22 μg/mg）， and reached peak level in August（♀＝2.49 μg/mg，♂＝2.78 μg/mg）. The average liver glycogen concentration in female and male snails was 1.09 and 0.89 μg/mg, respectively. The muscle glycogen concentration gradually decreased from February to June， the lowest was found in June （♀＝0.25 μg/mg，♂＝0.41 μg/mg）, and reached peak level in December （♀＝16.59 μg/mg，♂＝10.06 μg/mg）. The average muscle glycogen concentration in female and male snails was 7.99 and 6.05 μg/mg, respectively. There was a positive linear correlation between whole-body and liver glycogen concentrations（P＜0.05）, and both of them had the similar trend in their monthly change. A positive linear correlation was found among whole-body, liver and muscle glycogen concentrations（P＜0.05）.

Diagnosis and Treatment for Three Imported Plasmodium malariae Malaria Cases in Henan Province

DENG Yan，ZHOU Rui-min，ZHANG Hong-wei*，QIAN Dan，LIU Ying，CHEN Wei-qi，ZHAO Xu-dong

2014, 32(1):  18-61-63. 

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【Abstract】 Giemsa-stained blood film microscopy, CareStartTM rapid detection and PCR were used to detect the three cases who returned from Angola and Equatorial Guinea to Henan Province. Onset of malaria symptoms for two patients occurred 15 d and 27 d after their return from Angola, respectively. Two months after returning home, another case relapsed who had suffered from malaria in Equatorial Guinea. All three patients had the symptoms such as irregular fever, headache, chills and so on. Two cases had elevated total bilirubin and splenomegaly. The cases were confirmed as P. malariae infection by microscopic morphological examination. Amplified bands were produced by 18S rRNA nested PCR， which was the same with P. malariae in size, whereas the results of CareStartTM repaid detection test were all negative. They were cured by using artemisinin-based combination therapy （ACT）.

Identification of Mammalian Blood Meals in Anopheline Mosquitoes from Four Counties of Yunnan Province by Multiple PCR

ZHANG Jing-wei1，JIANG Jin-yong2，WANG Xue-zhong2，ZENG Xu-can2，ZHENG Yu-ting2，ZHOU Hong-ning1，2 *，LI Hong-bin3，LONG Zhi4，YANG Tai-zhuan5

2014, 32(1):  19-76-77. 

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 【Abstract】 From June to August 2012, the blood-sucking mosquitoes were captured around cattle-sheds and human houses in Yuanjiang County, Qiaojia County, Yongshan County, and Jinghong City of Yunan Province. Blood samples from mosquitoes were collected on filter paper. Multiplex PCR assay was used to detect the blood meal samples. Among the 145 mosquitoes captured, 123 were Anopheles sinensis（84.8%） and 22 A. minimus（15.2%）. Among the blood samples, corresponding bands were amplified in 134 samples. The result showed that the blood meals were from pigs （n=104）, cows （n=22）, dogs （n=4）, human （n=2）, cow and pig （n=1）, pig and human （n=1）. Human blood index of A. sinensis and A. minimus was 0.018 and 0.045, respectively.

Prevalence of Hydatid Disease in Baiyin City of Gansu in 2011

HE Yong-hu, QIANG Li-hong

2014, 32(1):  20-78-79. 

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【Abstract】 This study was performed to determine the prevalence of hydatid disease in Baiyin District, Pingchuan District, Jingyuan County, and Jingtai County of Baiyin City in Gansu Province during June-December, 2011. Echino-coccus infection in human was investigated by serological assay and type-B ultrasound examination. Sheep and dogs were dissected for pathological examination and their fecal samples were determined by ELISA. The prevalence of hydatid disease in human was 0.29%（52/17 699）. There was significant difference among different education levels, different districts, and age groups（P<0.05）. The sero-positive rate in children was 2.98%（198/7 376）. The infection rate in sheep was 2.83%（142/5 026）. The fecal antigen positive rate in dogs was 3.84%（73/1 902）.