Table of Content

30 August 2017, Volume 35 Issue 4

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In vitro induction of drug resistance of Plasmodium falciparum by an inhibitor of its dihydroorotate dehydrogenase

ZHOU Hong-chang*, ZHANG Hui, LI Xiao-yu, GUO Yue, YAO Yun-liang, WANG Sha

2017, 35(4):  2-317-321. 

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Objective To explore the effects of an inhibitor of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) (a dihydrothiophenone derivative, referred to as PfDHODH inhibitor 50) on cultured P. falciparum parasites of 3D7 (sensitive to chloroquine) and Dd2 strains (resistant to chloroquine), and investigate the underlying mechanisms. Methods The highly synchronized parasites were divided into three groups, negative control group, PfDHODH inhibitor 50 group at the ring stage and PfDHODH inhibitor 50 group at the trophozoite stage. The final concentration of the inhibitor was 80 nmol/L. Giemsa staining was performed at 0 (the ring stage), 24 (the ring stage) and 42 h after synchronization. Drug resistance was induced by gradually increasing the PfDHODH inhibitor 50 concentration. After three months the drug-resistant clone strains were obtained by limiting dilution. The half maximal inhibitory concentration (IC50) values of each strain in the presence of PfDHODH inhibitor 50, chloroquine or artemisinin were measured by the SYBR Green Ⅰ method. The Pfdhodh gene was amplified by PCR and sequenced to examine mutations. Results Compared to the control group, PfDHODH inhibitor 50 addition at the ring stage significantly inhibited the development of parasites from trophozoites to schizonts, while that added at the trophozoite stage induced obvious vacuolization and decreased density of cytoplasm and nucleus. Forty-four PfDHODH inhibitor 50-resistant strains were obtained, in which 24 strains were derived from Dd2 and 20 were derived from 3D7. Their IC50 values to PfDHODH inhibitor 50 were (2.284±0.096) and (0.678±0.018) μmol/L, respectively, which were increased by about 130 and 50 folds compared with their maternal strains Dd2 and 3D7. The IC50 values of Dd2-derived resistant strains to chloroquine and artemisinin were (0.011±0.002) and (0.014±0.004) μmol/L, respectively, and those of 3D7-derived resistant strains were (0.013±0.003) and (0.012±0.001) μmol/L, respectively. Compared with the maternal Dd2 strain, the IC50 of resistant strains to chloroquine decreased from (0.072±0.002) μmol/L to (0.011±0.002) μmol/L. Sequencing results revealed G181D point mutation in the PfDHODH protein sequence in 23 Dd2-derived resistant strains, G181D and K32N point mutations in 1 Dd2-derived resistant strain, while no PfDHODH mutation was found for 3D7-derived resistant strains. Conclusion PfDHODH inhibitor 50-resistant strains were obtained. The G181D mutation in PfDHODH may be the main molecular mechanism for the resistance to PfDHODH inhibitor 50.

NLRP3 inflammasome activation in liver is associated with the extent of hepatic fibrosis in mice infected with Schistosoma japonicum

WEN Zhen-cheng, JI Xiao-fang, LIN Gui-ying, TANG Juan-juan, WANG Man-ni, LIANG Cui-ying,

2017, 35(4):  3-322-326. 

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Objective To investigate the association of NOD-like receptor protein 3 (NLRP3) inflammasome activation with the extent of liver fibrosis in mice with Schistosoma japonicum infection. Methods Forty-eight SPF-grade female BALB/c mice at six to eight weeks of age were grouped as non-infection (n = 8), S. japonicum infection for 5, 6, 8 and 12 weeks (n = 10 in each). Each mouse in the infection group was infected with 20±3 cercariae through an abdominal transdermal patch. The liver tissues were collected for Sirius red staining to detect collagen deposition or liver fibrosis. SYBR Green quantitative PCR(qPCR) was performed to detect the relative mRNA expression of NOD-like receptor protein 3 (NLRP3), apoptosis associated speck-like protein containing a CARD domain (ASC), pro-caspase-1, pro-IL-1β and pro-IL-18. Immunohistochemical staining (IHC) and H-score were used to evaluate the protein levels of caspase-1 active fragment p10 and IL-1β. Results Sirius red staining revealed that in the S. japonicum infection group the collagen deposition area was the largest at 8 weeks post infection (29.66±1.07)%, followed by 6 weeks (21.69±1.24)%, 12 weeks (11.98±0.95)%, and 5 weeks (1.76±0.34)%. Compared with the non-infection group, qPCR results showed that at 5, 6, 8 and 12 weeks after infection, the relative mRNA expression of NLPR3 was 8.12±0.66 (P<0.01), 24.13±2.81 (P<0.01), 14.86±0.35 (P<0.01), and 6.74±0.67 (P<0.01), respectively; the relative mRNA expression of ASC was 0.82±0.14, 7.12±0.90, 3.08±0.87, and 4.13±0.93 (P<0.01); the relative mRNA expression of pro-caspase-1 was 1.14±0.72, 2.53±0.46 (P<0.05), 3.16±0.80 (P<0.01), and 2.19±0.87; and the relative mRNA expression of pro-IL-1β was 9.95±1.04, 117.76±10.01 (P<0.01), 36.98±11.73 (P<0.01), and 7.74±2.27. No significant changes were found for pro-IL-18 (2.42±0.36, 1.85±0.11, 1.74±0.10, and 1.69±0.15, respectively). The IHC H-scores of caspase-1 p10 at 5, 6, 8, and 12 weeks after infection were 2.80±0.12, 2.10±0.06, 8.57±1.00 (P<0.01), and 4.55±0.50 (P<0.01), while those of IL-1β were 0.13±0.13, 0.20±0.00, 3.30±0.59 (P<0.01), and 1.80±0.43, respectively. Conclusion S. japonicum infection for 8 weeks shows the most serious liver fibrosis. NLRP3 inflammasomes were significantly activated at weeks 6 and 8 post infection. NLRP3 inflammasome activation in the liver is positively correlated with the extent of hepatic fibrosis in infected mice.

Dynamic changes of density of Babesia microti in mice with latent infection after re-infection, immunosuppression, or random transmission to healthy mice

CAI Yu-chun1,2, CHEN Shao-hong2, LU Yan2, AI Lin2, YANG Chun-li2, CHEN Jia-xu2*

2017, 35(4):  4-327-332. 

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Objective To observe the infection dynamics of Babesia microti in mice with latent infection after re-infection, immunosuppression, or random transmission to healthy mice. Methods Twelve healthy BALB/c mice at the age of six weeks were intraperitoneally (i.p.) injected with 100 μl peripheral blood collected from infected mice (B. microti density, 20%) and then divided into 4 groups (control group, re-infection group, immunosuppression group and random transmission group, n = 3 in each). Tail blood collection was performed for 28 consecutive days to observe morphological changes of B. microti using Giemsa staining, calculate red blood cell (RBC) infection rate, and establish the mouse model of B. microti latent infection. Then the re-infection group once again received an i.p. injection of infected blood at the same dose as above and the immunosuppressive group received i.p. injection of dexamethasone (an immunosuppressant) for five consecutive days(0.5 mg/day). Orbital blood samples were collected in the random transmission group, which were then i.p. injected into 9 healthy BALB/c mice (1 ∶ 3). Tail blood was collected from the 3 groups of mice for consecutive 28 days to calculate RBC infection rate with microscopy and observe morphological changes of B. microti. Results B. microti parasites were first seen in RBC on day 3 in all the 4 groups. The RBC infection rate peaked on day 7(73.2%, 78.0%, 76.2% and 79.0% in control, re-infection, immunosuppression and random transmission groups, respectively), and gradually declined thereafter till the negative infection under a microscope, suggesting a latent infection phase. In the re-infection group, the rate of re-infection was 0 throughout the 28 days, while in the immune suppression group, re-infection was detected from day 2, reached a peak on day 12 (65.2%), and declined thereafter till 0 on day 22 (a latent infection phase). B. microti were firstly seen on day 4 in the transmitted nine mice, reached a peak on day 12 (35.0%-39.0%), and declined thereafter till the latent infection phase. The morphological changes of B. microti were basically similar among various groups. The early stage of infection was characterized by small ring bodies, while the peak stage was mainly composed of large ring bodies and filamentous bodies. Mmultiple parasitisms were also seen. Conclusion Premunition can be seen in mice with latent infection, which can be a source of infection. Immunosuppression of these mice may be followed by occurrences of similar density change dynamics as that observed after the first infection.

ChinaCloning, expression and potential immunodiagnostic evaluation of Echinococcus granulosus transaldolase

XIN Qi, JING Tao*, SONG Xiao-xia, GAO Hai-jun, SUN Xu-dong, LV Wei, Nabil Pervaiz, LU Jun

2017, 35(4):  5-333-341. 

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Objective To perform bioinformatics analysis of the gene encoding Echinococcus granulosus transaldolase(EgTAL), clone and express this gene, and investigate the role of EgTAL as a drug target for anti-hydatid treatment and the diagnostic value. Methods Multiple softwares were employed to analyze the physical and chemical properties, the conserved domain, phylogenetics, and tertiary structure of EgTAL. Egtal gene was amplified and cloned into vector pET30a. The constructed recombinant plasmid pET30a-Egtal was transformed into E.coli BL21 (DE3) for expression under the induction of IPTG. The expressed product was purified with His60 Ni Superflow affinity chromatography and identified by SDS-PAGE and Western blotting in the presence of serum of patient with cystic echinococcosis. The diagnostic value of EgTAL recombinant protein was assessed by ELISA in sera from 20 patients with cystic echinococcosis and from 20 healthy individuals. The enzyme activity of EgTAL was determined by spectrophotometry. Results The Egtal gene showed a length of 981 bp, encoding a 326-amino-acid protein with a predicted Mr of 36 332 and an isoelectric point (PI) of 5.11. The EgTAL protein contained a TAL-specific sequence DATTNPSLI (31-39 aa) and a key catalytic site. The analysis of phylogenetic tree revealed that the homology between EgTAL and human TAL was 62%. The tertiary structure of EgTAL had two complete protein chains, A and B. The pET30a-Egtal recombinant plasmid was constructed. The results of SDS-PAGE and Western blotting indicated that the recombinant protein EgTAL was highly expressed as a soluble protein in E. coli BL21(DE3) with an Mr of 36 332, and can be recognized by serum from patient with cystic echinococcosis. ELISA indicated that the average A450 value of sera from the cystic echinococcosis patients and healthy people were 1.189±0.0384 and 0.325±0.078, respectively, and 17 out of 20 serum samples from the patients showed positive results.The enzyme activity assay revealed that the purified EgTAL recombinant protein had a significant enzyme activity. At 30 min after addition of 60 μg EgTAL into the enzymatic reaction system, the A340 value dropped to 0.139±0.009 from 1.684±0.103.  Conclusion Egtal gene is cloned and recombinant protein EgTAL is highly expressed in E. coli BL21(DE3). The protein EgTAL shows an enzyme activity and potential immunodiagnostic value.

Study on the protease inhibitory activity of TtSerpin1, a serine protease inhibitor from Trichuris trichiura

SITU Yong-li1,2, SHAO Zheng1, DENG Li1, SUI Xi-xiang1, LI Hai-jian1,3, XU Qin-ying1,

2017, 35(4):  6-342-346. 

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Objective To express, identify and characterize the serine protease inhibitory activity of Trichuris trichiura serine protease inhibitor 1（TtSerpin1）. Methods The nucleotide sequence encoding for TtSerpin1 (GenBank No.MF401634) was amplified from adult T. trichiura cDNA and ligated into pET32a-sumo vector to construct the recombinant plasmid pET32a-sumo/TtSerpin1. The positive recombinant plasmid was transferred into Escherichia coli BL21 (DE3) and expressed with IPTG induction. After denaturation, refolding, purification by Ni-NTA resin affinity chromatography and cleaving fusion tag by SUMO protease on the resin, a single stage chromogenic assay was used to detect the inhibitory activity of rTtSerpin1 against serine proteases. Results The recombinant plasmid pET32a-sumo/TtSerpin1 was constructed successfully. The fusion protein expressed in E. coli was insoluble and became aggregated as the inclusion body. The purified TtSerpin1 after refolding showed inhibitory activity against serine proteases. At a concentration of 1 000 nmol/L, rTtSerpin1 inhibited the enzymatic activity of human cathepsin G (100 nmol/L), human neutrophil elastase (10 nmol/L), human proteinase 3 (200 nmol/L), porcine pancreatic elastase (10 nmol/L) and bovine pancreatic α-chymotrypsin (1 nmol/L) by approximately 60.89%, 82.84%, 21.21%, 58.32%, and 96.98%, respectively, but it showed a weaker inhibitory activity against human trypsin (1 nmol/L), porcine trypsin (1 nmol/L) and human fibrinolytic enzyme (5 nmol/L). rTtSerpin1 inhibited human cathepsin G and neutrophil elastase with a Ki value of (949.80±91.51) and (242.70±53.41) nmol/L, respectively. Conclusion TtSerpin1 shows an inhibitory activity against multiple serine proteases.

Expression and identification of microneme protein 2 of Toxoplama gondii in different prokaryotic expression strains

SUN Hui, LI Jin, ZHAO Jun, LIU Gong-zhen, YIN Kun, CUI Yong, XIAO Ting, XU Chao,

2017, 35(4):  7-347-350. 

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 Objective To express Toxoplasma gondii microneme protein 2(TgMIC2) in different Escherichia coli strains, and confirm its protein expression by Western blotting. Methods A primer pair was designed according to the TgMIC2 sequence in GenBank. The TgMIC2 gene was amplified by PCR using first strand cDNA as template after reverse transcription of total RNA from tachyzoites of T. gondii RH strain. The PCR products were digested with NdeⅠand Hind Ⅲ and cloned into the expression vector pET-30a(+). The recombinant plasmid was transformed into E. coli TOP10 and positive clones were confirmed by double restriction digestion and sequenced. The correct plasmid was transformed into E. coli strains BL21(DE3), ArcticExpress(DE3) and Shuffle, and then induced by IPTG for protein expression. The expressed proteins were purified with Ni-NTA affinity chromatography and analyzed by SDS-PAGE. The immune activity of the proteins was analyzed by Western blotting using His-tag monoclonal antibody as primary antibody. Results The TgMIC2 PCR product was 2 000 bp in length. SDS-PAGE analysis showed that TgMIC2 protein had a Mr of 80 000, and the expression pattern and amount of the recombinant protein differed among the strains. Soluble forms and inclusion bodies of recombinant TgMIC2 were seen in BL21 (DE3) and ArcticExpress (DE3) strains with the soluble protein consisting about 10% under different induction conditions(15 ℃, 16 h and 37 ℃, 4 h), while only inclusion body of recombinant TgMIC2 was seen in the Shuffle strain. Western blotting showed that both forms of TgMIC2 could be recognized by His-tag monoclonal antibody after purification. Conclusion pET30a-MIC2 plasmid is constructed and TgMIC2 is successfully expressed in different prokaryotic expression systems.

Cloning and expression of the full-length gene of group 8 allergen of Dermatophagoides farinae

WANG Nan1, TENG Fei-xiang1, YU Li-li1, YANG Li1, ZHANG Cheng-bo1, ZHOU Ying1,2, CUI Yu-bao1,2*

2017, 35(4):  8-351-356. 

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Objective To obtain the full-length gene of group 8 allergen of Dermatophagoides farinae, Der f 8, and construct a prokaryotic expression vector to express this gene. Methods Primers were designed according to the previously published partial sequence of Der f 8 (GenBank Accession No.AY283295) and synthesized. The Der f 8 fragment was amplified by RT-PCR using the total RNA of Dermatophagoides farinae as template. The full-length Der f 8 was obtained by rapid amplification of 5′ cDNA ends (RACE), ligated into pMD19-T vector and transformed into Escherichia coli. Positive colonies were selected for plasmid extraction followed by sequencing. Primers were designed based on the full sequence of Der f 8, and RT-PCR was performed using total RNA as template. The products were recycled from gel and incorporated into the pCold TF plasmid to construct the pCold TF-Der f 8 recombinant plasmid, which was transformed into E. coli, and incubated overnight. The positive colonies were used for plasmid extraction and sequencing. The pCold TF-Der f 8 plasmid was again transformed into E. coli BL21 for expression under the induction of IPTG. The expressed product was validated by SDS-PAGE. The physical and chemical properties, structure and function of Der f 8 were predicted by bioinformatics software. Phylogenetic tree was constructed. Results The Der f 8 fragment was amplified by RT-PCR with the product size of 600 bp. Sequencing result was as expected. The left part of full-length Der f 8 was obtained by 5′ RACE with a length of 300 bp, and confirmed by sequencing. The Der f 8 CDS region was amplified by RT-PCR using the primers designed based on the full-length Der f 8, with a length of 696 bp, and was confirmed by sequencing. SDS-PAGE showed that the target protein was expressed in a soluble form, with a relative molecular weight of 81 000. Bioinformatics analysis revealed a 98.49% homology between full-length Der f 8 and the reference sequence (GenBank No.AY283295). Functional analyses through ScanProsite, InterProScan and MotifScan identified glutathione S transferase activity of its protein, with its secondary structure comprising of alpha helixes (45.45%), an extended main strand(11.3%), and random coils (43.3%). The phylogenetic tree showed that Dermatophagoides farinae and Dermatophagoides pteronyssinus were clustered together. Conclusions The full-length Der f 8 cDNA has been obtained, and the prokaryotic expression vector has been constructed to express this gene.

Effect of soluble protein from Eimeria stiedai on a mouse model of subcutaneous colon tumor

YANG Gui-lian, HUANG Hai-bin, YANG Wen-tao, JIANG Yan-long,

2017, 35(4):  9-357-361. 

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Objective To investigate the effect of soluble protein from Eimeria stiedai(EsSP) on tumor growth, survival and immune status in tumor-bearing mice. Methods The minimum number of tumor cells that caused tumorigenesis was determined to establish a mouse model of subcutaneous colorectal cancer (CT26). EsSP was prepared from 108 E.stiedai sporulated oocysts by ultrasonic intermittent emulsification. One hundred and five BALB/c mice were randomly assigned into 7 groups, each receiving a subcutaneous injection of 5×105 CT26 cells at the right flank. Six experiment groups (A-F) were also injected intraperitoneally with different doses of EsSP (100.00, 50.00, 10.00, 1.00, 0.10, and 0.01 μg) once a day for 5 days, respectively. The control group were injected with the same volume of PBS. The tumor diameter was measured at 7, 11, 13, 15, 17, 19, 21, 23 days after inoculation, and the relative tumor volume and relative tumor proliferation rate (T/C) were calculated. Five mice in each group were sacrificed on day 25 after inoculation. The tumor was weighed and tumor inhibition rate was calculated. Orbital blood was collected to isolate peripheral lymphocytes. The effect of EsSP on the proliferation of lymphocytes in tumor-bearing mice was assessed by MTS colorimetric assay, and the stimulus index (SI) was calculated. CD4+/CD8+ T lymphocyte ratio in peripheral blood was determined by flow cytometry. The time and number of death were recorded for 80 days. Data were analyzed by one-way ANOVA. Results The minimum amount of tumor cells for tumorigenesis was 5 × 105 cells. At 23 days after inoculation, the tumor volumes in groups A, B and C were (435.2±41.1) mm3, (366.3±29.2) mm3, and (460.2±28.5) mm3, respectively, which were significantly smaller than (761.2±33.22) mm3, (810.4±38.36) mm3 and (865.2±35.29) mm3 in groups E and F and control group, respectively （P<0.05). The T/C values in groups A, B and C were (39.0±6.7)%, (33.3±8.9)%, and (35.0±8.1)%, all < 40%. The tumor weight in group B was (1.109±0.432) g, which was significantly lower than (1.946±0.289) g in the control (P<0.05), presenting with the highest tumor inhibitory rate of(43.0±14.6)%. MTS colorimetric assay showed that the SI values in groups B and C were (1.75±0.15) and (1.70±0.32), both significantly higher than the control [(1.38±0.18)] (P<0.05). Flow cytometry revealed that the ratios of CD4+/CD8+ T lymphocyte subsets in peripheral blood in groups A, B and C were (1.58±0.24), (1.74±0.22) and (1.61±0.16), respectively [P<0.01 or P<0.05, compared with (1.34±0.15) in the control]. The 80-day observation reported that 5 mice survived in groups B and C, while only 2 survived in the control group (P<0.05). Conclusions EsSP can inhibit tumor growth, improve the survival, change the tumor-induced immunosuppressive state, and enhance the anti-tumor immune response in tumor-bearing mice.
 

Production and identification of monoclonal antibodies against cockroach allergen Bla g 2

ZHANG Hao1*, MA Wen-jing2, HE Shao-heng3

2017, 35(4):  10-362-365. 

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Objective To produce anti-cockroach Blattella germanica allergen (Bla g 2) monoclonal antibody (mAb) and identify the specificity. Methods Six BLAB/c female mice were immunized with Bla g 2 protein every other week, for 4 times. Serum was obtained at 5 days after immunization, and an immune boosting was performed on the next day. On day 7, the mice were sacrificed and spleens were obtained to prepare the splenic cell suspension. The antiserum titer was determined and hybridoma cells were obtained by fusing the splenocytes with myeloma cells. The positive hybridoma cells were cloned by the limited dilution method. The specificity, titer, and subtype of monoclonal antibody were detected by Western blotting, indirect ELISA, and ELISA, respectively. Results The A450 value was > 0.100 when the mouse serum was diluted at 1 ∶ 16000, which suggested the successful immunization. Two hybridoma cell lines that were capable of stably secreting Bla g 2 mAbs were obtained, named Bla g 2-1 and Bla g 2-2. Western blotting demonstrated that both Bla g 2-1 and Bla g 2-2 mAbs specifically binded the Bla g 2 protein. The titers of mAbs from Bla g 2-1 and Bla g 2-2 were 1 ∶ 16 000 and 1 ∶ 8 000, respectively. ELISA results showed that both mAbs belonged to the IgG1 subtype. Conclusion Two monoclonal cell subtypes expressing Bla g 2 mAbs have been obtained, which can specifically bind the Bla g 2 protein.
 

Epidemiological investigation on hydatid disease/echinococcosis in Guoluo Tibetan Autonomous Prefecture in Qinghai Province

MA Xiao1, WANG Hu1, ZHANG Jing-xiao1, WANG Yong-shun1, CHENG Shi-lei1*, LIU Yu-fang1,

2017, 35(4):  11-366-370. 

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Objective To understand the prevalence of human and animal hydatid disease/echinococcosis in Guoluo Tibetan Autonomous Prefecture of Qinghai Province. Methods Sampling was made in counties of Banma, Dari, Gande, Jiuzhi, Maduo and Maqin in Guoluo Prefecture of Qinghai Province, 2-3 villages from each County. B ultrasonography was performed in residents at the age of > 1 year to calculate the prevalence of hydatid disease. ELISA was performed to detect serum anti-echinococcus antibody in humans, and visceral anatomy was used to detect hydatid cyst in murines and livestock. Canine fecal samples were collected in field to detect antigens with ELISA. Results A total of 15 890 residents received B ultrasonography, of whom 826 (5.20%) were positive for hydatid cysts. ELISA in 1 200 residents revealed a positive rate of 16.75% (201/1 200) for anti-echinococcus antibody. The prevalence of hydatid disease was highest in Dari County (11.93%, 430/3 605). The positive rate of serum antibody was highest in Gande County (48.15%, 65/135), followed by Banma County (12.77%, 136/1 065). The prevalence of hydatid disease and positive rate of serum antibody in females were 6.37% (499/7 839) and 26.01% (142/546) respectively, which were both significantly higher than those in males (4.06% and 9.02%). There was a relatively higher prevalence of hydatid disease in herdsmen, religious people, students and houseworkers (5.24%, 700/13 370; 6.46%, 41/635; 8.39%, 25/298; 5.68%, 18/317; respectively) among the occupations, and in age ranges of 60- years (6.91%, 76/1 100), 30- years (6.25%, 255/4 078) and ≥ 70 years (8.08%, 37/458). The serum positive rate was higher in age group of ≥ 70 years (48.28%, 14/29), 60- years (28.57%, 20/70), and 40- years (23.12%, 40/173). Among different educational levels, the prevalence of hydatid disease and positive rate of serum antibody were the highest in the illiterate population (5.85%, 431/7 365 and 62.61%, 72/115, respectively). Among populations with different living styles, the prevalence of hydatid disease and positive rate of serum antibody were the highest in those who settled in winter and move about in summer (6.36%, 642/10 087; 60.49%, 147/243). There were significant differences in different regions, genders, occupations, age groups, education levels and living styles (P<0.05). The positive rate of echinococcus infection in rodents was 2.41% (98/4 058) in Guoluo Prefecture; more specifically, it was highest in Jiuzhi County (4.82%, 49/1 016). The antigen positive rate in dog feces was 14.90% (303/2 034), and was the highest in Maqin County (18.76%, 160/753). The positive detection rate of hydatid cysts in yaks and sheep was 28.37% (570/2 009), and was the highest in Dari County (62.80%, 314/500). These were also significant differences among the regions in positive rates of yaks, sheep, murines and dogs (P<0.05). Conclusion The echinococcosis epidemicity is still serious in Guoluo. Dogs remain a major source of transmission.

The world list of ticks. 2. Ixodinae（Acari ∶ Ixodida ∶ Ixodidae）

CHEN Ze1*, WEN Ting-huan2

2017, 35(4):  12-371-381. 

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This paper compiled a list of world species in subfamily Ixodinae (Ixodidae) alphabetically. Towards the end of 2015, altogether 247 valid species names of Ixodinae accommodated in one single genus have been consented by most of the world ixodologists. So far, 24 Ixodes tick species have been recorded in China. In order to facilitate the scientific exchange between international and domestic colleagues on ticks and tick-borne diseases, a latest list of world Ixodinae valid names was introduced. Meanwhile a translated Chinese term for each valid species or named genus was proposed in a succinct and systematic way. Furthermore, a series of abbreviations for the nominated genera and subgenera of Ixodinae were provided.

Interpretations of terms used in toxoplasmosis and clinical implications of serodiagnosis and treatment strategy

SHEN Ji-long*

2017, 35(4):  13-382-386. 

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Toxoplasma infection in immunocompetent individuals is usually asymptomatic with chronic infection. But in immunocompromised individuals including patients with HIV/AIDS, the reactivation of latent infection may cause severe even fatal complications. Acute Toxoplasma infection in pregnant women may result in serious adverse outcomes. This review tries to interpret several terms used in the WS/T 486-2015 Diagnosis of Toxoplasmosis standard and the laboratory test indicators. It also introduces the laboratory diagnosis of toxoplasmosis in pregnancy in other countries and the treatment schemes, so as to provide reference for toxoplasmosis prevention and treatment in China.

Discussion on the insufficiency of Criteria for Diagnosis of Toxoplasmosis (WS/T 486-2015) and treatment of #br# Toxoplasma gondii infection in pregnancy

HUANG Yi-xin*, DENG Yong, ZHOU Yong-hua, TONG De-sheng

2017, 35(4):  14-387-389. 

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In this paper, we discuss the insufficiency of Criteria for Diagnosis of Toxoplasmosis (WS/T 486-2015) and propose some modifications to be made in future version. We also discuss the treatment of Toxoplasma gondii infection in pregnancy, including the measures for eliminating Toxoplasma gondii infection and indicators for recommendation of artificial termination of pregnancy. This is aimed to stimulate discussion in the field and promote prenatal and postnatal care.

Reply to “Discussion on the insufficiency of Criteria for Diagnosis of Toxoplasmosis (WS/T 486-2015) and treatment of #br# Toxoplasma gondii infection in pregnancy”

WEN li-yong1*, XIONG Yan-hong2, YAN Xiao-lan1, ZHENG Bin2, GUAN Ya-yi2,

2017, 35(4):  15-390-395. 

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In this article, we aimed to address the previously-raised issues of insufficiency of Criteria for Diagnosis of Toxoplasmosis (WS/T 486-2015) and the treatment of Toxoplasma gondii infection in pregnancy, in three aspects, the hygienic standard classification, the diagnosis of Toxoplasma infection, and the treatment of toxoplasmosis in pregnancy. By doing this we hope to further promote the publicity and implementation of the Criteria for the Diagnosis of Toxoplasmosis.

Post kala-azar dermal leishmaniasis

GUAN Li-ren*, GAO Chun-hua

2017, 35(4):  16-396-401. 

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Post kala-azar dermal leishmaniasis (PKDL) is rather common in endemic areas of visceral leishmaniasis (kala-azar) in India and Sudan, and is the main infectious source of local visceral leishmaniasis. Despite the low incidence of PKDL in China, it still occurs in kala-azar endemic areas in the western part of the country. The similarity between PKDL-induced skin nodules and the tumer-form leprosy is very likely to cause misdiagnosis and mistherapy of the two diseases. In this review, we focus on research progress on the clinical manifestation, diagnosis, treatment, and pathogenesis of PKDL, as well as proposing key points to distinguish between PKDL and cutaneous leishmaniasis, in the aim to help professionals engaged in research and control of leishmaniasis and skin diseases.

SWOT analysis on the standard formulation in the #br# control of parasitic diseases

XIONG Yan-hong, ZHENG Bin*, ZHOU Xiao-nong

2017, 35(4):  17-338-341. 

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The study analyzes the strength, weakness, opportunity and threat of the standards of parasitic diseases by SWOT analysis comprehensively, and it puts forward some development strategies specifically, in order to provide indicative references for the further development.
 

SWOT analysis of malaria elimination program in Yunnan Province

ZENG Xu-can, ZHOU Zi-you, ZHOU Xing-wu, LI Jian-xiong, LUO Chun-hai,

2017, 35(4):  18-392-395. 

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The Chinese government has made a commitment to eliminate malaria by 2020. However, there are still autochthonous malaria cases in border areas of Yunnan Province with intense population flow. Therefore, malaria elimination by 2020 is rather challenging in the province. Here, we analyze the strengths, weaknesses, opportunities and threats (SWOT) of the action of malaria elimination program carried out by the Yunnan institute of parasitic diseases(YIPD), and raise some targeting strategies accordingly, in order to provide reference for effective elimination of malaria in Yunnan Province.

Schistosoma japonicum: hemozoin formation and its biological significance

SUN Jun*

2017, 35(4):  19-402-406. 

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By summarizing the characteristics of morphology and structure, formation and degradation of Schistosoma japonicum hemozoin in its gut, this review proposes that the formation of S. japonicum hemozoin is not the way to detoxify toxic heme from metabolisim of hemoglobin, but rather serves to transport iron to eggs to meet the need for iron during egg development. Current understanding of the morphology and formation as well as biological significance of S. japonicum hemozoin are also summarized.

Research progress on iron metabolism in Schistosoma spp.

ZHAO Jin-ying1,2, LI Yan-wei1, WANG Shi-ping2

2017, 35(4):  20-407-411. 

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The treatment of schistosomiasis has been relying on the mere use of praziquantel. However, the concern of drug resistance caused by long-term use has urged the development of new drugs against schistosomes. There is a solid evidence that iron plays a critical role in the development and reproduction of schistosomes. Hence, it may be a potential effective way to control schistosomiasis by designing a therapeutic target based on the iron metabolism of schistosomes. This paper reviews the recent studies on iron metabolism of schistosomes, in order to provide theoretical reference for the development of new drugs and therapeutics.
 

In vitro cultivation of ethrythrocytic-stage Plasmodium yoelii

WANG Bing-li*, HUANG Jia-fu, CHEN Fan

2017, 35(4):  21-412-414. 

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The persistent in vitro cultivation of ethrythrocytic-stage Plasmodium yoelii has not come to successful results. In this study, we modified the cultivation based on Trager’s method. The modifications included: placing P. yoelii parasites in a small chamber of a suspension system, and agitating them into suspension by a magnetic stirrer; daily replacement of the culture medium and addition of new reticulocytes into the medium; and cultivation at 37 ℃ with 5% CO2. The in vitro cultures were injected daily into mice through tail vein, and the infection status in mice was examined on day 3. Results showed that the proportion of red blood cells with parasite infection increased to 2.200% within 24 h of in vitro cultivation, then decreased till 0% on day 10. As to the mouse model, the mice all showed positive infection from days 1 to 9, with highest rate on day 1 (54.960%), and no infection on day 10. This result suggested that the cultivation of erythrocytic-stage P. yoelii can be maintained up to 9 days in vitro without losing infectivity.
 

Analysis of imported malaria cases in Daiyue District of Tai′an City during 2008-2016

AN Xing-gui1, MA Yuan-qin2, ZHANG Lin-lin1

2017, 35(4):  22-415-416. 

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A descriptive review was made on 111 imported cases of malaria in Daiyue District of Tai′an City filed through the online reporting system from 2008 to 2016, consisting of 76 cases of falciparum malaria (68.5%), 15 cases of plasmodium vivax(13.5%), 7 cases of quartan vivax (6.3%), 9 cases of ovale malaria (8.1%), and 4 unclassified cases (3.6%). The cases distributed throughout the months, predominated by November, December and January (34, 30.6%). They returned mainly from Africa(99.1%). All were males, and 79.3% were among 30-49 years old. The time from onset to diagnosis ranged 5 hours to up to 42 days, with an average of 5.4 days. All got recovered after treatment except one case of falciparum malaria who died in 2011.