Table of Content

28 February 2017, Volume 35 Issue 1

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ORIGINAL ARTICLES

Polymorphism analysis of the block 5 region in merozoite surface protein-1 gene of imported and local Plasmodium vivax in Yunnan Province

Ying DONG, Ai-ming SUN, Meng-ni CHEN, Yan-chun XU, Xiang-hua MAO, Yan DENG

2017, 35(1):  1-7. 

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Objective To analyze the sequence of Plasmodium vivax merozoite surface protein-1 (PvMSP-1) and allele polymorphism in imported and local vivax malaria parasites in Yunnan Province. Methods Blood samples on filter paper were collected from imported and local vivax malaria cases in Yunnan Province during August 2012 and September 2015 and information of epidemiological history was recorded. Plasmodium DNA was extracted by a DNA extraction kit, and the block 5 region in PvMSP-1 gene was amplified by PCR. The PCR products were sequenced and blasted with reference sequences M75674, AAN86237, M60807, ABJ53045, AAN86238 and BAA18977. The sequence polymorphism in block 5 region of PvMSP-1 was analyzed with MEGA 5.04 and Arlequin3.5.1 softwares. The conserved sites, genetic distances among sequences and Shannon-wiener index among alleles were calculated. The clustering tree was drawn according to the genetic distances between the amino-acid sequences. Results A total of 847 blood samples were collected from the malaria cases, comprising of 61 samples from local cases, 66 from imported cases from Africa, and 720 from Myanmar. The block 5 region in PvMSP-1 was successfully amplified in 278 samples, and sequencing was successfully made in 206 of them. The peptide coded by the block 5 region had a length of 193 to 222 aa. The amino acid sequence alignment showed that in 206 samples the proportion of genotypes of Sal-1, Belem and Recombine was 59.2% (122/206), 23.3% （48/206） and 17.5% （36/206）, respectively. The proportion of Sal-1 genotype in imported cases from Myanmar and Africa and in local cases was 58.8% (104/177), 73.3% (11/15) and 50% (7/14), respectively. The genotypes Sal-1, Belem and Recombine had 51, 9 and 6 different alleles. The 66 alleles had a Shannon Wiener index (H’) of 0.955 and an expected heterozygosis (He) of 0.567. The 206 DNA sequences had a 665-bp homologous locus, comprising of 75 conserved sites (11.3%, 75/665) and 590 variable sites (88.7%, 590/665). The genetic distances between sequences were all less than 0.4. The clustering analysis showed that the 206 sequences were clustered into two categories with three branches. The homology of Recombine with Belem genotype was 91%-92%, higher than with Sal-1 genotype (82%-83%). Conclusion The block 5 region in PvMSP-1 gene from local and imported Plasmodium vivax in Yunnan Province has varied forms of alleles, and the Sal-1 genotype is predominant among the three genotypes.

The regulatory effect of dendritic cells on Th17 cell differentiation and function in mouse infected with Plasmodium yoelii

Guang CHEN, Lei LIU, Fang-fang WANG, Sheng BI, Lan LUO, Ju-xiang SU, Hui-ming ZHANG, Lian-shun CAI, Zi-lin GONG

2017, 35(1):  8-12. 

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Objective To investigate the regulatory effect of dendritic cells (DCs) on Th17 cell differentiation and function during mouse infection with Plasmodium yoelii 17XL strain (Py17XL) and the underlying mechanisms. Methods Twelve female BALB/c mice were randomly assigned into the infection group (Py17XL), the TLR4 blocking group (Py17XL + TLR4), TLR9 blocking group (Py17XL + TLR9), and TLR4 and TLR9 combined blocking group (Py17XL + TLR4+TLR9)(n=3 in each group). Mice in the Py17XL + TLR4 or the Py17XL + TLR9 group received intraperitoneal injection of 10 μg anti-TLR4 or 50 μg anti-TLR9 antibody (both 0.4 ml) to block DCs function at one day before infection. The Py17XL group received same volume of PBS. All groups were then given intraperitoneal injection of 1×10⁶ red blood cells (RBCs) infected with Py17XL. The RBC infection rate was calculated on days 0, 3 and 5 after infection, and spleen cell suspension was prepared, in which the CD11c⁺TLR9⁺ and Th17 cells were counted by flow cytometry. The levels of IFN-γ and IL-10 in supernatant of spleen cell culture were determined by ELISA. Results Flow cytometry showed that DCs were successfully blocked. On day 5 after infection, 28%, 29%, 31% and 16.3% mice showed parasitemia in the Py17XL group, the Py17XL + TLR4 group, the Py17XL + TLR9 group, and the Py17XL + TLR4 + TLR9 group, respectively, and on day 7, the proportions were 43.3%, 47.5%, 32.5% and 8%. Mice in the Py17XL group and the Py17XL + TLR4 group all died, while those in other groups began to die from day 6. There was a slow rise of parasitemia rate in the Py17XL + TLR9 group and the Py17XL + TLR4 + TLR9 group compared with the Py17XL group, with a significant extension of survival to days 11 and 15. Results of flow cytometry showed that the proportions of Th17 cells were 1.2% and 1.44% in the Py17XL + TLR9 group and the Py17XL + TLR4 + TLR9 group on day 5, both sighificantly decreased compared with the Py17XL group (1.9%)(P < 0.05, P < 0.01). ELISA revealed that the levels of IFN-γ and IL-10 on day 5 in the Py17XL + TLR4 + TLR9 group [(232.4 ± 15.5） pg/ml and（1 791.2 ± 58.2） pg/ml, respectively] were significantly higher than those in the Py17XL group[(90.7 ± 50.1) pg/ml and (962.6 ± 409.0) pg/ml](P < 0.05, P < 0.01). Conclusions The differentiation and function of Th17 cells are regulated by DCs during Py17XL infection. Blockade of DCs decreases parasitemia and extends lifetime of mice. Further studies are needed to clarify the exact mechanisms.

Polymorphisms of tryptophan-rich antigen gene of Plasmodium ovale subspecies from imported malaria cases in Henan Province

Rui-min ZHOU, Su-hua LI, Ya-lan ZHANG, Dan QIAN, Cheng-yun YANG, Ying LIU, Yu-ling ZHAO, Hong-wei ZHANG, Bian-li XU

2017, 35(1):  13-17. 

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Objective To analyze the genetic sequence of tryptophan-rich antigen (PoTRA) gene of Plasmodium ovale subspecies from imported malaria cases in Henan Province in 2015. Methods Blood samples were collected from 22 imported ovale malaria cases in Henan Province in 2015. After DNA extraction, PoTRA was amplified by nested PCR, and was inserted into the pMD18-T vector. The plasmid was extracted and sequenced, and the results were blasted in GenBank to determine the subspecies of P. ovale. The sizes and species of the PoTRA gene were analyzed. The amino-acid sequence of PoTRA was also aligned to analyze the difference in amino acid sequence. The phylogenetic tree was constructed to analyze the genetic relationship among the samples by neighbor-joining. Results Of the 22 imported cases, eight (36.4%) were infected with P. ovale wallikeri, which had two sizes, the predominant 245 and 299 bp. The other 14 cases (63.6%) were infected with P. ovale curtisi, which had three sizes, the predominant 299, 317 and 335 bp. Amino-acid sequence alignment revealed that the two types of P. ovale wallikeri differed in two amino-acid units, MANPINMANPIN and AITPIN, while the three types of P. ovale curtisi differed in amino-acid units TITPIS and TINPIN. The phylogenetic tree showed that the 22 samples belonged to two subpopulations of P. ovale curtisi and P. ovale wallikeri, wherein the P. ovale curtisi was further divided into two sub-branches, and samples with sizes of 317 and 335 bp were in the same sub-branch with a closer genetic relationship. Conclusion Two subspecies, P. ovale curtisi and P. ovale wallikeri, are identified from the imported ovale malaria cases in Henan Province in 2015. The P. ovale curtisi has three genetic types and P. ovale wallikeri has two genetic types of PoTRA gene, revealing genetic polymorphisms of PoTRA.

The effect of Dermatophagoides pteronyssinus group 2 allergen T cell fusion epitope on STAT6 signaling in mice with asthma

Xiao-dong ZHAN, Bin-bin DUAN, Ning TAO, Chao-pin LI

2017, 35(1):  19-23. 

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Objective To investigate the alterations of signal transducer and activator of transcription factor 6（STAT6） signaling in a mouse model of asthma receiving treatment with Dermatophagoides pteronyssinus group 2 allergen（Der p 2） T cell fusion epitope and the mechanisms of the specific immunotherapy. Methods Thirty mice were randomly divided into three groups by the random number table method: the asthma group, the treatment group receiving immunotherapy with Der p 2 T cell fusion epitope, and the negative control group （PBS group）（n = 10 in each group）. Mice in the asthma and the treatment groups received intraperitoneal （i.p.） injection of 100 μl Der p 2 solution [PBS containing 100 μg/ml Der p 2 and 2% Al（OH）₃] on days 0, 7 and 14, respetively, while mice in the PBS group received same volume of PBS containing 2% Al（OH）₃. From day 21, 30-min steam inhalation of 0.5 μg/ml Der p 2 was applied to the asthma and treatment groups （once daily for 7 successive days）, and the PBS group inhaled same volume of PBS. From day 25 to day 27, the mice in the treatment group received i.p. injection of 200 μl of Der p 2 T cell fusion epitope （100 μg/ml） while the PBS and the asthma groups received the same volume of PBS. Mice were sacrificed at 24 h after the last inhalation, the bronchoalveolar lavage fluid （BALF） collected, and the total protein was extracted from the lung tissue. The levels of IFN-γ, IL-4 and IL-13 in BALF were determined by ELISA. The expression of STAT6 and phosphorylated STAT6 （p-STAT6） in the lung tissue was detected by Western blotting. Data were analyzed with the one-way variance analysis （ANOVA） method. Results The level of IFN-γ in the treatment group[（234.40 ± 24.46） pg/ml] was significantly higher than that in the asthma group[（155.80 ± 20.53） pg/ml]（P < 0.01）. The levels of IL-4 and IL-13 in the treatment group [（30.00 ± 5.50） pg/ml and （174.50 ± 25.99） pg/ml, respectively] were both significantly lower than those in the asthma group[（53.28 ± 8.26） pg/ml and （308.10 ± 28.32） pg/ml, respectively]（P < 0.01）. Similarly, the levels of STAT6 and p-STAT6 in the treatment group（0.803 ± 0.221 and 0.966 ± 0.323, respectively） were both significantly lower than those in the asthma group （1.669 ± 0.296 and 1.735 ± 0.298, respectively）（P < 0.01）. Conclusion The Der p 2 T cell fusion epitope may function through inhibiting STAT6 to treat asthma in mice.

Component analysis of excretory/secretory protein from Trichinella spiralis adult worm

Xiao-di YANG, Zhi-yong TAO, Yang CHENG, Qi WU, Xiao-li WANG, Di SONG, Lan-song XU, Ren-min XUE, Xue-lian CHANG, Hui Zhang, Rui WANG, Xing-zhi CHEN, Qiang FANG

2017, 35(1):  24-29. 

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Objective To analyze the component of adult worm excretory/secretory protein（AWESP） from Trichinella spiralis using the shotgun method, and find out the active component underlying its regulatory effect on colitis in humans. Methods The T. spiralis AWESP was prepared, separated by SDS-PAGE, lysed with trypsin, and analyzed by shotgun LC-MS/MS. The protein components were determined with the Masco software and classified using the Gene Ontology（GO） method in cellular components, molecular functions, and biological processes. Results The AWESPs isolated by SDS-PAGE had a M_r of 15 000-116 000. A total of 280 proteins were revealed by LC-MS/MS, of which 96 were identified by Masco software, 98 were putative, and the remaining 86 were unclear. Preliminary results showed that 4 proteins had regulatory potential for colitis, including cysteine protease inhibitor, serine protease, 53 000 excretory/secretory antigen, and glutathione-S-transferase. GO enrichment analysis showed that the identified proteins had 104 different molecular functions, involved in 363 biological processes. Conclusion As revealed by the Masco software, T. spiralis AWESP has complex components and 96 have been identified in this study. Four of them are preliminarily shown to be associated with the anti-colitis effect of T. spiralis.

Discovery of Culex inatomii（Diptera ∶ Culicidae）in Chongming, Shanghai

Yuan FANG, Wen-qi SHI, Yi ZHANG, Qiu-an HU, Zheng-bin ZHOU, Jia-tong WU, Liang-liang ZHANG

2017, 35(1):  31-35. 

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Objective To record the discovery of Culex inatomii in Chongming, Shanghai. Methods Larvae and adult mosquitos of Cx. inatomii were collected in Dongtan of Chongming Island from May to November in 2015 and 2016, and their morphological characteristics were observed. The genomic DNA was extracted from adult mosquitos, PCR was performed to amplify the cytochrome c oxidaseⅠ(COⅠ) gene. Multiple alignment of COⅠ sequence was conducted with ClustalW2. Pairwise distances within and between species were calculated using MEGA v5.10 based on COⅠ sequences of Cx. inatomii, Cx. modestus, Cx. pipiens pallens, and Cx. tritaeniorhynchus. The phylogenetic tree of the above four species was constructed using neighbor joining, maximum likelihood, and Bayesian methods. Results One hundred and fifty-six adult female mosquitos and 36 larvae of Cx. inatomii were collected. Larvae were reared to adult stage in the laboratory (17 female, 19 male). Morphologically, the subapical lobe of the sidepiece in male genitalia was divided into two parts, the anterior part having 2 bladed setae, and the posterior part having 1 bladed setae and 1 lanceolar strong setae. This strucutre can be used to distinguish Cx. inatomii from Cx. modestus. PCR of COⅠ resulted in products of approximately 650 bp. They were sequenced and the sequencing result was submitted to GenBank (accession number, KX555565-KX555570). Multiple sequence alignment revealed a 96% sequence similarity of COⅠ between Cx. inatomii and Cx. modestus. The genetic distance between Cx. inatomii and Cx. modestus was 0.047, and that within them each was 0.003 and 0.011, respectively. The phylogenetic tree showed that the four species clustered as a monophyletic clade, and each formed an individual lineage. Cx. inatomii had a closer relationship with Cx. modestus, while distant from the other two species. Conclusion We recorded the discovery of Cx. inatomii in Chongming, Shanghai.

The effect of leptin transgenic Plasmodium yoelii on mouse body weight

Kai LI, Hong ZHENG, Feng ZHU, Yong FU, Wen-yue XU

2017, 35(1):  36-41. 

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Objective To investigate the effect of leptin transgenic Plasmodium yoelii on mouse body weight. Methods To construct the leptin gene-containing CRISPR/Cas9 recombinant plasmid which had the 5′UTR and 3′UTR of MIF（macrophage migration inhibitory factor） of Plasmodium yoelli 17XNL strain at two ends, the exogenous mouse leptin gene was inserted downstream of MIF coding region through homologous recombination, resulting in the PYC-MIF-Leptin recombinant plasmid. The recombinant plasmid was then electroporated into P.y 17XNL mature schizonts, and the transgenic schizonts were used to infect a Kunming mouse via tail vein injection. The trangenic P.y clone was screened by pyrimethamine selection and identified by PCR. The trangenic or wild-type P.y was used to infect a C57BL/6 mouse respectively. Blood sample was collected through eye ball and tail vein, and immunofluorescence and RT-PCR were performed to determine the expression of leptin protein in the parasites. Finally, PBS （200 μl） containing trangenic or wild-type P.y （1 × 10⁴） was injected through the tail vein into C57BL/6 mice（n = 5 respectively）. The negative control received a same volume of PBS. The changes of parasitemia and body weight were recorded every two days. Results The leptin-expressing recombinant plasmid PYC-MIF-Leptin was constructed successfully. Results of DNA sequencing of transgenic parasites confirmed the integration of leptin gene at the downstream of MIF gene and successful transcription. Immunofluorescence results indicated successful expression of mouse leptin protein. The weight loss was significant in mice infected with transgenic parasites on day 17（17.26 ± 1.40）g, decreased by 10.7%, but not in the other two groups. Both transgenic and wild-type parasites began to decline when parasitemia reached about 10%, but the transgenic parasites proliferated more rapidly. Both disappeared at 23 days. Conclusion Infection with leptin transgenic parasites decreases the body weight of the infected mice.

Proteomics analysis of Paragonimus skrjabini

An-mei LI, Ma-li WU, Yu-ting HUANG, Zhi-lai GUO

2017, 35(1):  43-47. 

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Objective To perform a proteomics analysis for metacercariae, juvenile and adult worms of Paragonimus skrjabini. Methods Crabs were collected in P. skrjabini endemic areas of Kaiyang and Bijie in Guizhou Province. Metacercariae were isolated, placed in PBS, and were used to infect three SD rats（10 metacercariae/rat） by intragastric administration and infect three male dogs（20 metacercariae/dog） through feeding. The rats were sacrificed at 1 month after infection to obtain juvenile worms. The dogs were sacrificed at 3 months after infection to obtain adult worms. The metacercariae, juvenile and adult worms were lysed, and total protein was extracted by ultrasonication. The total protein content was determined by Bradford method and separated by SDS-PAGE and two dimensional gel electrophoresis. Images of two dimensional gel electrophoresis were analyzed using the PDquest 8.0 software. The dots with difference were digested and analyzed with mass spectrometry. Finally, online searches in NCBI and local databases were performed. Results Results of SDS-PAGE showed that the total protein of metacercariae, juvenile and adult worms was concentrated within M_r of 25 000-116 000. Fifty-one protein dots with difference were found by two dimensional gel electrophoresis, comprising of 20 dots for metacercariae, 25 for juvenile worms and 6 for adult worms. Thirty-six peptide sequences of metacercariae and juvenile worms were analyzed. They were basically determined to be Achrornobacter lyticus protease Ⅰ（a lysine-specific serine protease）, ascorbate reductase protein, glutathione s-transferase DHAR2 analogue, heat shock proteins Hsp82 and Hsp96-β, actin, cystatin, etc., by online searches, and cysteine, actin and heat shock protein by local searches in the diginea database（downloaded to a local computer from NCBI）. Mass spectrometry was not performed for adult worms, as the variation of grayscale value between their spots was far less than those for metacercariae and juvenile worms. Conclusion One difference is that the metacercariae of P. skrjabini have actin, while the juvenile worms have detoxification proteins and stress proteins. But they both have hydrolases and cysteine enzymes.

Cloning of galectin-1 gene of Angiostrongylus cantonensis and testing the agglutination property of the galectin-1 protein

Xing-pan LI, Meng-jing ZHAO, Xiao-meng SHI, Lan-zhu YAN, Bao-long YAN, Hui-cong HUANG

2017, 35(1):  48-52. 

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Objective To clone and express the galectin-1 gene of Angiostrongylus cantonensis, and test the agglutination property of its protein. Methods The three-dimensional structure of galectin-1 was analyzed with Swiss Model. Total RNA was extracted from male worms of A. cantonensis. Primers were designed for galectin-1 based on its coding region (GenBank Accession No. JN133961.1). RT-PCR was performed, and the PCR products were subcloned to pCold Ⅲ plasmid and transduced into Escherichia coli BL21 strain. The recombinant plasmid was extracted from positive clones on LB plate containing 100 μg/ml Kanamycin, and validated with double digestion, PCR identification and sequencing. The confirmed positive clones of E. coli BL21 with the recombinant plasmid were grown in LB medium containing ampicillin (100 μg/ml, 100 μl). IPTG was added to induce expression of the plasmid. The galectin-1 recombinant protein was purified with Ni-NTA beads, and analyzed with SDS-PAGE and Western blotting using anti-serum of mouse immunized with whole worms of A. cantonensis. The agglutination reaction with red blood cells in fresh blood of ICR mouse was observed for the 10-fold serial dilutions of recombinant proteins (5.55 × 10^-1-5.55 × 10^-5 ng/μl). Results The Swiss Model analysis showed that the functional galectin-1 had a non-dimeric form. As was expected, the RT-PCR products had a size of 850 bp. Results of double digestion, PCR and sequencing showed successful construction of the pCold Ⅲ-galectin-1 plasmid. SDS-PAGE revealed expression of soluble recombinant fusion protein with molecular weight of ~36 000. Western blotting showed that the galectin-1 protein was recognized by mouse anti-serum. In addition, the minimun concentration of galectin-1 that showed significant agglutination reactions with mouse red blood cells was 5.55 × 10^-4 ng/μl. Conclusion The galectin-1 clone can be expressed in the pCold Ⅲ plasmid, and its protein product has agglutination property.

Analysis on the application of three methods for malaria diagnosis

Li JIANG, Zhen-yu WANG, Yao-guang ZHANG, Min ZHU, Xiao-ping ZHANG, Xiao-jiang MA, Yan-yan HE, Qian ZHU, Shou-fu JIANG, Li CAI

2017, 35(1):  53-58. 

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Objective To test the usage of microscopic examination, antigen detection（rapid dignostic test, RDT） and nucleic acid test（PCR） for detection of malaria cases. Methods The blood test results for malaria and suspected malaria cases during 2012-2015 were retrospectively reviewed. Taking the confirmed cases as a gold standard, the three methods were compared in aspects of diagnosis indices, specificity of identification species, and cost effectiveness. Results A total of 212 samples were included, each analyzed with the three methods. Based on the results of the three tests, 167 （78.8%） were determined to be positive for malaria, and 45 negative （21.2%）. Of the positive samples, 120 （71.9%） were infected with Plasmodium falciparum, 22 （13.2%） with P. vivax, 17（10.2%） with P. ovale, 6 （3.6%） with P. malariae, and 2 （1.2%） with mixed infections. The method of PCR had the highest diagnostic efficiency （96.2%, 204/212）, followed by RDT （93.2%, 192/206; P > 0.05 vs. PCR） and the microscopic method （88.2%, 187/212; P < 0.05 vs. RDT and PCR）. Similarly, the PCR method had the highest overall coincidence rate to the confirmed cases （95.3%, 202/212）, followed by RDT （93.2%, 192/206） and microscopy （88.2%, 187/212; P < 0.05 vs. PCR）. As to the identification specificity among species, the PCR method（95.6%, 43/45） was superior to microscopy （91.1%, 41/45; P > 0.05 vs. PCR） and RDT （68.9%, 31/45; P < 0.05 vs. PCR）. As to the identification of a particular species （P. falciparum）, RDT performed best （100%, 116/116）, followed by PCR （93.3%, 112/120） and microscopy （84.2%, 101/120）. Based on the comprehensive evaluation on 14 indicators including if it is a diagnostic criterion, equipment and technical requirement, diagnostic performance, time cost, and the need of technical training and promotion, we found that the RDT method had the highest score（37 of 42）, while microscopy and PCR were scored 26 and 27, respectively. Conclusion Under the falciparum malaria-dominated epidemiological situation, PCR and RDT show a higher detection efficiency, PCR and microscopy perform better in species identification, and RDT has the highest cost-effectiveness.

Mouse immune responses elicited by eukaryotic expression plasmid and prokaryotically expressed protein of Brugia malayi myosin 29 epitope

Yu-ye XIA, Qian XU, Ting-ting SUN, Hao FANG, Wei LIU, Shi-juan LU, Zheng FANG

2017, 35(1):  59-63. 

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Objective To determine the immune responses elicited in BALB/c mice by vaccination with eukaryotic expression plasmid pcDNA3.1（+）-BmM29 containing Brugia malayi myosin 29（BmM29） epitode and prokaryotically expressed recombinant BmM2 protein（rBmM29） respectively. Methods rBmM29 protein was expressed in E. coli strain BL21, purified as recombinant protein vaccine, and administered via multiple subcutaneous injections. The purified recombinant plasmid pcDNA3.1（+）-BmM29 was used as the nucleic acid vaccine and injected into the tibialis anterior muscle. Sixty BALB/c mice were randomized to receive three immunizations（with intervals of 2 weeks） with PBS （100 μg, group A）, pcDNA3.1（+）/CpG （100 μg/30 μg, group B）, pcDNA3.1(+)-BmM29/CpG （100 μg/30 μg, group C）, rBmM29/CpG（50 μg/30 μg, group D）, or pcDNA3.1（+）-BmM29/rBmM29/CpG （two injections of pcDNA3.1（+）-BmM29/CpG 100 μg/30 μg followed by a rBmM29/CpG 50 μg/30 μg）. Serum was prepared through ophthalmectomy at week 4, 6, and 8 after primary immunization, and the serum IgG titer was determined by ELISA. The mice were sacrificed at week 8, splenocyte suspension cultured for 48 h, and levels of INF-γ and IL-4 in the supernatant detected by ELISA. Results ELISA results showed that the A₄₉₀ values of serum IgG in groups A-E were 0.038 ± 0.050, 0.053 ± 0.009, 0.360 ± 0.035, 0.456 ± 0.025, 0.370 ± 0.025 at week 4, 0.045 ± 0.003, 0.045 ± 0.005, 0.510 ± 0.018, 0.548 ± 0.010, 0.552 ± 0.018 at week 6, and 0.041 ± 0.004, 0.044 ± 0.009, 0.606 ± 0.047, 0.674 ± 0.042, 0.770 ± 0.041 at week 8, significantly higher in groups C, D and E than in groups A and B （P < 0.05） at all time points, and significantly higher in group E than in groups C and D（P < 0.05） at week 8. The IFN-γ levels in splenocyte culture supernatant at week 8 after primary immunization were （47.72 ± 8.94）, （50.43 ± 2.81）, （304.78 ± 8.42）, （242.28 ± 5.99）, and（426.52 ± 6.76） pg/ml in groups A-E, respectively, significantly higher in groups C-D than in groups A and B（P < 0.05）, and in group E than in groups C and D（P < 0.05）. The IL-4 levels in splenocyte culture supernatant were（60.00 ± 11.14）, （57.71 ± 15.95）, （93.17 ± 12.56）, （96.67 ± 11.48）, and （101.17 ± 5.81） pg/ml, significantly higher in groups C-D than in groups A and B（P < 0.05）. Conclusion Both the recombinant plasmid pcDNA3.19(+)-BmM29 and rBmM29 protein could elicit specific humoral and cellular immune responses in mice. Combined immunization with nucleic acid vaccine and protein vaccine is superior to each of the two alone.

Investigation and genetic identification on Babesia infection in rodents in some areas of Fujian Province

Fang-zhen XIAO, Xiu-qing Peng, Guo-ying XU, Yang CHEN, Dai-hua LIN, Yan-qin DENG

2017, 35(1):  63-67. 

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Objective To explore the status of Babesia infection in rodents and the genetic characteristics of Babesia spp. in Fujian Province. Methods Rodents were captured by the night trapping method in Shaowu, Qingliu, Shunchang, Yong’an, Changle and Youxi during 2014-2015. The rodent species was identified, and information on the time and place of capture, species and sex of rodents was recorded. Blood samples was collected, in which the fragment of 18S rRNA gene of Babesia spp. was amplified by PCR. The PCR products were sequenced and the phylogenetic tree was constructed for homology analysis. Data on positive rate were analyzed with Chi-square or Fisher exact test. Results Two hundred and nine rats were captured, comprising of 71 domestic and 138 wild rats. The overall positive rate was 9.6% （20/209）. The positive rate in domestic rats was 2.8% （2/71）, including one Rattus norvebicus and one Rattus flavipectus. The positive rate in wild rats was 13.0% （18/138）, including 13 Bandicota indica, one Rattus losea, 2 Rattus confucianus and 2 Rattus fulvescens. The positive rate was significantly higher in wild rats than in domestic rats （P < 0.05）. The Youxi region had the highest positive rate（14.9%, 13/87）, followed by Yong’an（13.6%, 3/22）, and no positive rat was found in Qingliu. The positive rate in the male rats was 7.9% （9/114）, and that in the females was 11.6% （11/95）. The positive rate was highest in adult rats （10.4%, 18/173）, followed by young ones （6.3%, 2/32）. No positive rat was found in old rats. There was no significant difference in positive rate among different regions, between male and female rats, or among different ages （P > 0.05）. The sequences of PCR products had a 100% homology. The BLAST results revealed the species to be Babesia microti. The phylogenetic tree showed that the sample sequence was the most homologous with Babesia microti from Zhejiang Province（GenBank Accession No: JQ609305）. Conclusion There occurs Babesia microti infection in rats in part areas of Fujian Province. The positive rate was higher in wild rats than in domestic rats.

Analysis of schistosomiasis prevalence in endemic areas using retrospective space-time permutation scan statistics

Yu-wan HAO, Jing-bo XUE, Jun-fang XU, Fei HU, Yun ZHANG, Chun-li CAO, Yao RUAN, Tian TIAN, Li-juan ZHANG, Jing XU, Dan-dan LIN, Yi DONG, Shi-zhu LI

2017, 35(1):  68-0. 

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Objective To analyze the characteristics of schistosomasis prevalence by using the spatial epidemiological method, and test the application of retrospective space-time permutation scan statistics in determining mountainous and lake-type endemic areas of schistosomiasis. Methods The data of schistosomasis in humans, cattle and snails in Jiangxi Province during 2009-2014 and in Yunnan Province during 2004-2013 were collected and analyzed. The temporal and spatial distribution of schistosomiasis endemic areas in the two provinces was analyzed with retrospective space-time permutation scan statistics. Results The prevalence of schistosomiasis in residents and Oncomelania snails showed a trend of decline in Jiangxi, from 0.21% and 0.03% in 2009 to 0.01% and zero in 2014. A similar trend was found in cattle, from 1.25% in 2012 to 0.12% in 2014. The average annual percentage change (APC) in residents was -47.36% (P < 0.05). The space-time permutation clustering analysis revealed a temporal and spatial clustering of schistosomiasis prevalence from 2009 to 2014 in residents, cattle, and snails, with 3, 2 and 1 clustering areas, respectively, all distributed in Poyang Lake Region. A similar declining trend of schistosomiasis prevalence was found in residents, snails and cattle in Yunnan during 2004-2013, from 2.49%, 0.70% and 3.76% in 2004 to no infection in residents and snails and 0.02% in cattle in 2013. The APC in residents was -49.17% (P < 0.05). There was a temporal and spatial clustering of schistosomiasis prevalence during 2004-2013 in residents, cattle, and snails, with 2, 2 and 6 clustering areas, respectively. Conclusion A declining trend of schistosomiasis prevalence is shown in lake-type endemic areas in Jiangxi during 2009-2014 and in mountainous endemic areas in Yunnan during 2004-2013. The retrospective space-time permutation scan statistics reveal a clustering of schistosomiasis in humans, cattle, and snails, suggesting its applicability in analyzing the temporal and spatial distribution of schistosomiasis.

Epidemiological characteristics of imported malaria in Sichuan Province in 2015

Li LI, Yang LIU, Guo-jun XU, Tao YU, Yan ZOU, Xiao-hong WU, Bo ZHONG

2017, 35(1):  75-79. 

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Objective To analyze the epidemiological characteristics of imported malaria in Sichuan Province in 2015 in the aim of providing scientific basis for malaria control. Methods The epidemiological data of malaria in Sichuan Province reported through the Infectious Disease Reporting and Management System of China Information System for Disease Control and Prevention in 2015 were collected and analyzed. Results In 2015, 290 malaria cases were reported in Sichuan Provinc, consisting of 158 falciparum malaria cases （54.5%）, 107 vivax malaria cases （36.9%）, 14 ovale malaria cases （4.8%）, one quartan malaria case（0.3%）, and 10 mixed infections of vivax malaria and falciparum malaria （3.5%）. Five cases of falciparum malaria died. The reported cases were all imported, with a major source of Africa （271, 93.4%）, in which Ethiopia （83） and Angola （49） were two major sources. The cases were reported continuously from January to December, with the majority（139, 47.9%） being reported in December, August, June and July. The cases distributed mainly in Guangan, Chengdu, Nanchong, Mianyang, Deyang, Luzhou and Suining（243, 83.8%）. Among the 209 cases who first visited medical units after onset, 118 cases were diagnosed as malaria, the misdiagnosis rate at first visit was 43.5% (91/209). Conclusion The malaria cases reported in Sichuan Province in 2015 are all imported from overseas, mainly infected with P. falciparum and P. vivax, and imported mostly from Africa. There is a high rate of misdiagnosis in medical units in Sichuan Province.

A retrospective analysis on the diagnosis and reporting of imported malaria in Jiangxi Province during 2012-2015

Lei LEI, Zhi-gui XIA, Zhi-hong LI, Yan-feng GONG, Ning XIAO

2017, 35(1):  80-84. 

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Objective To provide scientific basis for malaria surveillance in the elimination phase by retrospectively analyzing the diagnosis and reporting of imported malaria in Jiangxi Province. Methods Data on malaria endemic situation and individual cases during 2012-2015 were collected through the National Information Management System for Infectious Diseases and the Report and Information Management System for Parasitic Diseases Control and Prevention. Detailed information on primary medical units, laboratory testing units, reporting units, diagnostic methods, time from onset to first medical visit, time from first medical visit to reporting, and time from onset to reporting was analyzed with the descriptive analysis method. Results A total of 207 malaria cases were reported during 2012-2015 in Jiangxi, all were imported cases and 96.62% （200/207） were diagnosed with laboratory tests. The main primary medical units were found to be county-level （29.95%, 62/207） and prefecture-level （25.60%, 53/207） medical institutions, while the main laboratory testing units were prefecture-level medical institutions（35.27%, 73/207） and county-level CDCs （20.29%, 42/207）. There was a significant difference in the proportion of different laboratory testing units among the years（P < 0.05）. The median time from onset to first medical visit was 1 d （0-149 d）, from first medical visit to reporting was 3 d （0-144 d）, and from onset to reporting was 5 d （0-149 d）. Conclusions The first visit and the laboratory testing of malaria cases mainly occur in the prefecture-level and county-level medical institutions.

Diagnosis and treatment of a local case of Taenia saginata infection in Zhejiang Province

Yan FENG, Wei RUAN, Xin-jin LOU, Li-nong YAO

2017, 35(1):  85-88. 

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Objective To diagnose and provide treatment for a local case of taenia infection in Zhejiang Province and identify the species of the worm. Methods The information of disease onset, clinical feature and therapeutic process was collected and epidemiological investigation was carried out. The anal cellophane swab was used to detect the eggs. Areca and pumpkin seeds were used for deworming. Morphological observation, PCR amplification and sequencing of cytochrome C oxidase 1 （COX1） gene were performed for the discharged worm. Results The epidemiological results showed that the patient did not go outside Pujiang County in the past two years, and had no history of eating raw pork, beef or animal offal. But she often had barbecues and hot-pot food, occasionally with raw vegetables. Taenia eggs were found on her perianal skin. The discharged worm was suspected to be Taenia saginata or Taenia asiatica by morphological observation. PCR amplification of COX1 resulted in a band of 832 bp, which was 99%, 96% and 88% homologous to COX1 of Taenia saginata （GenBank accession number: AB107239.1）, Taenia asiatica（GenBank accession number: AB107235.1） and Taenia solium（GenBank accession number: AB066485.1）, respectively. Conclusion According to the clinical feature, epidemiological information and sequencing results, this case is confirmed to be a local infection of Taenia saginata.

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The role of Th17/Treg imbalance in liver fibrosis in schistosomiasis

Yong-hua ZHOU, Ying-ying YANG, Xiao-lin FAN, Xue SAI

2017, 35(1):  89-93. 

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Liver fibrosis in schistosomiasis is a serious pathological consequence from immune reactions to schistosome infection. The progression of liver fibrosis depends on the state of immune response. Recent studies have found that Th17 and Treg cells are two subsets of CD4⁺T cells. The Th17 cells are mainly involved in inflammatory responses, while the Treg cells mainly mediate downregulation of the responses. Under normal conditions, the differentiations of the two subsets are inhibited by each other, and they function oppositely. The balance between Th17 and Treg cells, as well as the balance between them, play an important role in the maintenance of homeostasis and are involved in inflammatory responses, tissue trauma, fibrosis and development of many diseases. This paper reviews the role of Th17/Treg cells and their imbalance in liver fibrosis in schistosomiasis.

Research progress on application of DNA barcoding technique in Culicidae taxonomy

Yu-yan GUO, Lei LUO, Xue-li ZHENG

2017, 35(1):  93-98. 

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DNA barcoding technique is a fast and accurate method for species identification. The DNA barcoding based on cytochrome c oxidase subunit Ⅰ (COⅠ) has recently been successfully applied for species identification of Culicidae. In this paper, we introduce the technique and principle of DNA barcoding, application and limitation of the technique based on CO I gene for species identification, as well as research development on applications of other molecular makers such as COⅡ, 16S RNA, and the first and the second internal transcribed spacers (ITS1 and ITS2) in species identification and to assist the COⅠ gene in identifying Culicidae species.