Table of Content

30 December 2016, Volume 34 Issue 6

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Malaria Situation in the People’s Republic of China in 2015

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

2016, 34(6):  1-477-481. 

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The 2015 malaria epidemiological data reported through the annual malaria statistics reporting system were collected and analyzed. Totally 3 288 malaria cases were reported in 664 counties of 31 Provinces/Municipalities/Autonomous Regions （P/M/A） in 2015, which increased by 6.8% in comparison to that of 2014 （3 078 cases）, and the incidence in 2015 was 0.024 0/10 000. The cases were reported primarily from Provinces of Yunnan （18.4%, 606/3 288）, Jiangsu （12.3%, 405/3 288）, Sichuan （8.8%, 290/3 288）, Guangxi （7.2%, 236/3 288） and Shandong（6.4%, 212/3 288）. Of all the cases, 40（1.2%, 40/3 288） were indigenous cases, mainly distributed in the border area of Yunnan （six counties）, Tibet （one county）, Liaoning （one county） and Hainan （one county）. There was one case of whom the source of infection was unknown. The locally-infected falciparum malaria was only found in Cangyuan County of Yunnan（1 case）. The prevalence of indigenous malaria in Motuo County of the Tibet Autonomous Region was over 1/10 000. Meanwhile, there were 3 248（98.8%, 3 248/3 288） abroad-imported cases which widely distributed in the 31 P/M/As. In addition, 3 265（99.3%, 3 265/3 288） of the reported cases were confirmed in reference laboratories, comprising 878 cases of Plasmodium vivax（26.9%, 878/3 265） 1 992 cases of  P. falciparum（61.0%, 1 992/3 265）, 76 cases of P. malariae（2.3%, 76/3 265）, 272 cases of P. ovale（8.3%, 272/3 265） and 47 cases of mixed infection（1.4%, 47/3 265）. Furthermore, 163 cases（5.0%, 163/3 288） with severe clinical symptoms were reported in 14 P/M/As, with 20 deaths（0.6%, 20/3 288） in 10 P/M/As. Totally 3 116 malaria cases were reported through the China Information System for Disease Control and Prevention, including 39 indigenous cases. These data reflect achievements in malaria elimination, despite that challenges remain in boarder areas of Yunnan Province and in Motuo County of the Tibet Autonomous Region. Efforts are still needed in risk assesment for malaria re-transmission.

Detection of Genetic Mutations Associated with Drug Resistance in Imported Plasmodium falciparum

XU Chao, WEI Qing-kuan，LI Jin，XIAO Ting，YIN Kun，WANG Yong-bin，KONG Xiang-li，XU Yan, CUI Yong，SUN Hui，ZHU Song，YAN Ge，HUANG Bing-cheng*

2016, 34(6):  2-482-488. 

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 Objective To investigate the mutation of genes associated with drug resistance（Pfcrt, Pfmdr1, Pfdhfr and K13） in imported Plasmodium falciparum in Shandong Province. Methods Blood was collected from 94 falciparum malaria cases who returned from Africa in 2014. Genomic DNA for P. falciparum was extracted from the blood samples and nested PCR was performed using primers specifically designed for Pfcrt, Pfmdr1, Pfdhfr and K13. The PCR products were sequenced. Gene mutations were analyzed by sequence alignment. Results The 94 imported cases were from 18 African countries. Nested PCR was successful on DNA from all the blood samples except for Pfcrt amplification in one sample. Sequence analysis revealed three types of mutations Pfcrt K76T （36.6%, 34/93）, Pfmdr1 N86Y （21.3%, 20/94）, and Pfdhfr S108N （98.9%, 93/94） （χ2=127.5， P<0.05）. K13 C580Y mutation was not found. Co-occurrence of K76T, N86Y, and S108N was found in 6 blood samples （6.5%）, which were imported from Liberia（2）, Angola（1）, Equatorial guinea（1）, Congo（1）, and Guinea（1）. Co-occurrence of K76T and S108N mutations was found in 28 samples（30.1%）, and that of N86Y and S108N in 14 samples （15.1%）. Forty-four samples（47.3%） harbored S108N mutation only, and one sample was null for any of the mutations. Conclusion There are mutations in Pfcrt, Pfmdr1, and Pfdhfr in imported Plasmodium falciparum in Shandong Province. No mutation was found for the K13 gene.

Mutations of Plasmodium falciparum Multidrug Resistance 1 Gene in Imported Plasmodium falciparum in Wuhan

JIA Xi-shuai, ZHOU Shui-mao*, XU Ming-xing, YANG Yan, WU Kai

2016, 34(6):  3-489-492. 

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Objective To identify Plasmodium falciparum multidrug resistance 1（Pfmdr1） point mutations in imported Plasmodium falciparum in Wuhan. Methods Blood samples were collected from returnees infected with P. falciparum in endemic areas of Africa and Myanmar during 2010-2015 in Wuhan City. Nested PCR primers were specifically designed for Pfmdr1 gene loci 86, 1042 and 1246 of P. falciparum. The Pfmdr1 gene was then amplified by nested PCR, and the products were digested by restriction enzyme ApoⅠ, AseⅠ and EcoRⅤ, respectively. The mutation rate for loci 86, 1042 and 1246 was analyzed. Results A total of 187 patients with falciparum malaria  were involved in the study. Pfmdr1 was amplified from all the blood samples. Restriction enzyme digestion revealed mutation rate of 19.3%（36/187）, 4.3% （8/187） and 9.6%（18/187） for loci 86, 1042 and 1246, respectively. In detail, the mutation rate for loci 86, 1042 and 1246 was 20.6%（36/175）, 2.9%（5/175） and 10.3%（18/175） respectively in the 175 samples from Africa, and only 3 cases with locus 1042 mutation were found in the 12 samples from Myanmar. Conclusion The loci 86, 1042 and 1246 mutations of Pfmdr1 have all been found in the samples from Africa, with only one point mutation （locus 1042） found in samples from Myanmar.

Sequencing and Phylogenetic Tree Construction of 18S Ribosomal DNA from Five Species of Plasmodium from Yunnan Border between China and Myanmar and Other Areas

CHEN Mu-xin1,2, CHEN Jia-xu2, LIU Wei2, FENG Xin-yu2, CHEN Shen-bo1,2,CHEN Shao-hong2, CAI Yu-chun1,2, XU Bin2, HU Wei1,2*

2016, 34(6):  4-493-499. 

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Objective To analyze sequence variation and construct phylogenetic tree based on 18S ribosomal DNA among five species of Plasmodium in Yunnan border between China and Myanmar and other areas. Methods Blood samples（or DNA samples)from malaria patients were collected from 2000 to 2015 in Yunnan border and Myanmar and other areas. DNA was extracted from blood samples, and the 18S rDNA fragment was amplified, sequenced and aligned with relevant sequences available in the GenBank. The phylogenetic tree was constructed by methods of neighbor joining（NJ）, maximum likelihood（ML）, and maximum parsimony（MP）, respectively. Results A total of 94 blood samples or DNA from malaria patients were collected. The 18S rDNA was successfully amplified from all the samples. Sequence alignment revealed variations of 0-0.2%, 0-0.1%, 0-0.1%, 0-0.1% and 0 for 18S rDNA sequence among Plasmodium falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi, respectively. The phylogenetic tree constructed with the three method showed consistency. Phylogenic analysis revealed that there were five big branches of Plasmodium spp. studied. The P. falciparum branch clustered with the isolates from Cameroon（KC428741, KC428742）, Brazil（KC906718）, and Malaysia（HQ283221） in GenBank. The P. vivax branch clustered with isolates from Cameroon（HF945443）, India （HM014361, JQ627158）, and Colombia （U83877）. However, the samples Pv11, Pv18 and Pv21 formed a small branch that showed closer phylogenetic relationship with P. cynomolgi（L07559）, an isolate from Macaca fascicularis. Moreover, P. malariae samples from Yunnan Province including Pm1, Pm3 and Pm4 clustered to form a small branch, and then clustered with samples from Hainan Province, showing geographical diversity. All the isolates of P. ovale clustered with isolates from Vietnam（EU935736 and AF387038）. All the isolates of P. knowlesi clustered into a branch, and showed close relationship with those from Myanmar（GU816250 and GU816246）. Conclusion There is no significant difference in 18S rDNA gene of the five species of Plasmodium from Yunnan border between China and Myanmar and other areas. The phylogenetic tree constructed with the NJ, MP and ML methods shows consistency.

Exploration of Using One-step Reverse Transcription PCR in Detection of Four Species of Human Malaria Parasites

LI Mei1，WANG Zhen-yu2，ZHANG Tao3，XIA Zhi-gui1*

2016, 34(6):  5-500-505. 

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Objective To explore the application and specificity of one-step reverse transcription PCR（RT-PCR）in detecting four species of human Plasmodium parasites. Methods Blood samples were collected from a falciparum malaria case, a vivax malaria case, an ovale malaria case, and five quartan malaria cases. RNA and DNA were isolated. One-step RT-PCR and one-step real-time RT-PCR were performed on the RNA digested with DNase to amplify the Plasmodium 18S rRNA. Traditional PCR and one-step RT-PCR were used to amplify 18S rRNAs and 18S rDNAs in  differentially diluted RNAs（with or without DNase digestion） and DNAs. The lowest detectable dilution concentrations for the two amplification systems were compared. Results One-step RT-PCR produced specific bands of 310, 394 and 323 bp, which were sequenced to be 18S rRNA of P. falciparum, P. ovale, and P. vivax. No specific band for P. malariare was found. The one-step real-time RT-PCR results showed fluorescence for all the four species, and all had a melting curve with a single peak except for P. malariare. The lowest detectable dilution concentration by one-step RT-PCR varied from 1 to 10-4 based on the DNA or RNA template amount. Specifically, the lowest detectable dilution concentration of DNA was similar to or lower than that of original RNA by an order of magnititude, and both were lower than that of DNase-digested RNA. Further, the sensitivity of one-step RT-PCR evaluated in terms of lowest detectable dilution concentration was 10-1 000 times higher than that of the traditional PCR. Conclusions The one-step RT-PCR technique can be applied in the detection of P. falciparum, P. ovale, and P. vivax in fresh blood samples. But its use in detecting P. malariae parasites needs further evaluation.

Influence of Age on the Susceptibility of  Anopheles stephensi to Plasmodium berghei Infection

SONG Xiu-mei, WANG Jing-wen*

2016, 34(6):  6-508-512. 

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Objective To investigate the ability of Anopheles stephensi at different ages to transmit Plasmodium berghei and elucidate the possible mechanisms. Methods To study and compare the susceptability of A. stephensi of different ages to P. berghei, 4-day and 25-day female A. stephensi were fed with P. berghei infected BALB/c mouse blood with parasitaemia 4%-8%. At 8 days after infection, the mosquitoes were dissected and the number of intestinal Plasmodium oocysts was counted under microscope. Difference in the susceptability to Plasmodium infection was analyzed between the two age groups. To study the intestinal bacteria load in A. stephensi, LB plate culture was used to detect the intestinal bacteria in the mosquitoes of the two groups before infection, and real-time quantitative PCR（qPCR） was performed to check the culturable bacteria and the total bacteria load. The expression levels of major immune response effectors cecropin（CEC1, CEC3）, defensin（DEF）, gambicin（GAM）, attacin（ATT）, nitric oxide synthase（NOS）, dual oxidase（DUOX）, and thioester protein 1（TEP1） in 4-day and 25-day mosquitoes were determined by qPCR. Results At 8 days after infection, the median of oocyst number in 4-day mosquitoes was 139, which was nearly 46 times of that in 25-day mosquitoes（median, 3）（P<0.01）. There was a significant difference in the intestinal bacteria load between 4-day and 25-day mosquitoes. The results of qPCR showed that the total bacteria load in 25-day mosquitoes was 1.5 times of that in 4-day mosquitoes（P<0.05）. By LB plate culture, proliferation of 28 889 colony forming units（cfu） bacteria was found for 25-day mosquitoes, which was 9 times of that for 4-day mosquitoes（3 200 cfu）（P<0.05）. In addition, the NOS expression level in 25-day mosquitoes was 2.4 times of that in 4-day mosquitoes（P<0.01）, while the expression levels of antimicrobiota peptides ATT, DEF, CEC3, and CEC1 in 25-day mosquitoes were only 27%, 48%, 14%, and 61% of those in 4-day mosquitoes, respectively（P<0.05）. The expression levels of GAM, DUOX, TEP1 showed no difference between the two groups. Conclusion The antimicrobiota peptides in A. stephensi are significantly downregulated with age increase, together with increased intestinal bacteria load and NOS expression, resulting in enhanced resistance to Plasmodium.

Diagnosis and Treatment of the First Imported Case of Plasmodium knowlesi Infection in China

PAN Bo1, PEI Fu-quan1, RUAN Cai-wen1, LIN Rong-xing1, CEN Yong-zhen1, LIU Meng-ran1, DENG Zhuo-hui1, REN Wen-feng1, LIAO Yin-bin1, LI Xiao-heng4*

2016, 34(6):  7-513-516. 

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Objective To diagnose and treat the first imported active case of Plasmodium knowlesi infection in China. Methods The clinical information of the patient was collected. Microscopy of blood smear was conducted after Giemsa staining. Genomic DNA was extracted from blood, and PCR was conducted to amplify rDNA. The PCR products were sequenced and analyzed with BLAST. Results The patient returned from a one-week tour in a tropical rain forest in Malaysia. The first disease attack occurred in Guangzhou on Oct. 16, 2014, with fever, shivering and sweating. The patient was initially diagnosed as malaria and hospitalized on Oct. 26, 2014. Microscopic observation revealed typical forms of P. knowlesi in blood smear. The red blood cells became enlarged, with big trophozoites appearing as a ring with dual cores and dark brown malaria pigment. The trophozoites were slightly bigger and thicker than P. falciparum. The schizont had 6-8 merozoites, with obvious brown malaria pigment. PCR resulted in a specific band of 1 099 bp. BLAST analysis showed that the sequence of the PCR product was 99% homologous to P. knowlesi（acession No. AM910985.1, L07560.1 and AY580317.1）. The patient was diagnosed as P. knowlesi infection, and was then given an 8-day treatment with chloroquine and primaquine, together with dihydroartemisinin piperaquine phosphate tablet. The patient was discharged after recovery on Oct. 28, 2014. Conclusion According to the clinical symptoms, epidemiological history and laboratory test, the patient has been confirmed as P. knowlesi infection. It may also be the first active case of knowlesi malaria reported in China.

Expression and Detection of Lentivirus-mediated Green Fluorescent Protein in Schistosoma japonicum

XIN Yue1，ZHAO Nan1，LI Qing1*，HU Wei1,2

2016, 34(6):  8-517-521. 

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Objective To test if the green fluorescent protein gene can be expressed in Schistosoma japonicum and produce green fluorescence signals there. Methods The spontaneous fluorescence in Schistosoma japonicum at different developmental stages was observed by fluorescence microscopy. The cultured 14-day schistosomula were infected with the lentiviral vector containing cyto megalo virus（CMV）-driven zsGreen gene, or were incubated with RPMI 1640 medium containing 10% fetal bovine serum as a negative control. At 48 h after infection, genomic DNA and total RNA were extracted, and cDNA was synthesized. PCR was performed to detect the genomic integration of zsGreen gene and the expression of zsGreen mRNA. Green fluorescence was observed under a fluorescence microscope at 24 h and 48 h after infection, and at various time points after replacement with fresh culture medium. Results There was no obvious spontaneous fluorescence in schistosomula, but the adult worms showed clear spontaneous fluorescence. PCR results showed a specific band of 173 bp from the schistosomula genomic DNA, which corresponded to the zsGreen gene. Gene sequencing also confirmed this result. At 24 h after infection, green fluorescence was seen in the intestine of schistosomula, which was, however, suspected to be food residues. At 48 h after infection, evenly-distributed green fluorescence emerged across the intestine, and the fluorescence was brighter than the background fluorescence of the lentiviral culture medium. The fluorescence weakened on day 3 after replacement with fresh 1640 medium, and disappeared a week later. The green fluorescence re-emerged 2 weeks after replacement with the lentivirus-containing 1640 medium. No fluorescence was seen in the negative control group. Conclusion The exogenous green fluorescent protein gene can be expressed in Schistosoma japonicum. However, the green fluorescence seen under the fluorescence microscope needs further verification.

Risk Assessment of Snail Output Via the Mud Balls of Transplanted Seedlings in a Nursery Stock Park in Middle Region of Zhejiang Province

XIE Juan1, WEN Li-yong1*, ZHU Kuang-ji2, YAN Xiao-lan1, JIANG Neng-ming3,LIN Li-jun1, SHAO Feng-yao2,ZHANG Jian-feng1, YU Li-ling1, DU Hai-juan1

2016, 34(6):  9-522-526. 

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Objective To investigate the distribution of snails in a nursery stock park in the middle region of Zhejiang Province and assess the risk of snail output via the mud balls of transplanted seedlings, to provide scientific data for making strategies for snail control. Methods We selected three species of seedlings including Osmanthus fragrans（a large tree）, Camellia sasanqua（a small tree）, and Purpus privet（a type of shrub） in a nursery stock park in a snail-positive middle region of Zhejiang Province during 2014-2016 to calculate the areas of regions with snails and the density of living snails. In 30 trees of each species, the distribution of snails within the seedlings ground diameter（radius of investigation, 100 cm for Osmanthus fragrans; 30 cm for Camellia Sasanqua and Purpus Privet） and in different soil layers（surface and superficial layers, 0-3 cm; deep layer, 3-10 cm） was assessed. In addition, the presence of snails in mud balls of 50 trees of Photinia fraseri（a small tree with high density of snails） was investigated to assess the risk of snail output. Results In the planting areas of Osmanthus fragrans（3 930 m²）, Camellia sasanqua（2 000 m²）, and Purpus privet （1 700 m²）, the areas of snail-positive regions were 200, 900 and 800 m2, respectively, with the density of living snails being 0.08, 0.56 and 0.55/0.1 m2. For Osmanthus fragrans, Camellia sasanqua and Purpus privet, 238, 654 and 645 snails were detected respectively within their seedlings ground diameter, including 159（66.8%）, 461（70.5%） and 376 （58.3%） snails in the surface layer, respectively, which were significantly higher than those in the superficial and deep layers（P＜0.01）. Snails were found in all the 50 trees of Photinia fraseri（3 726 snails, 706 adult snails and 3 020 immature snails, 75 snails/tree on average）. Conclusion There is a high density of snails in the nursery stock park in the middle region of Zhejiang Province. The snails are distributed mainly in the surface layer, suggesting a risk of snail output through mud balls.

Toxoplasma gondii Rhoptry Protein 17 Inhibits the Apoptosis of Mouse Macrophages via Activation of Activator Protein 1 Signaling

WANG Hai-long*, XING Jia-xin, GUO Shan, LI Jia-jie, LIU Hong-li, MENG Xiao-li, LIU Juan-juan, ZHAO Rui-jun, YIN Guo-rong

2016, 34(6):  10-529-533. 

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Objective To investigate the effect of Toxoplasma gondii rhoptry protein 17（ROP17） on γ-interferon（IFN-γ）-induced apoptosis of mouse J774A.1 monocyte macrophages. Methods The J774A.1 cells were transfected with recombinant plasmid p3×Flag-CMV-14/TgROP17 or empty plasmid p3×Flag-CMV-14. After addition of IFN-γ, flow cytometry and Western blotting were performed to detect apoptosis and the protein levels of phosphorylated c-Jun and apoptosis-related proteins cleaved Caspase-3, Bcl-2, Bcl-xL and Bcl-3. The p3×Flag-CMV-14/TgROP17 plasmid and c-Jun shRNA were co-transfected into J774A.1 cells, after which IFN-γ was added to induce cell apoptosis. The levels of cleaved Caspase-3 and Bcl-3 were analyzed using Western blotting. Results Flow cytometry showed that the apoptosis rate of cells overexpressing ROP17［（3.73±0.51）%］ was significantly lower than that of the control cells［（7.78±1.10）%, P<0.05］. Western blotting showed significant differences in protein levels of phosphorylated c-Jun（0.196±0.028 vs. 0.075±0.010）, Bcl-3（0.461±0.063 vs. 0.108±0.013） and cleaved Caspase 3（0.015±0.004 vs. 0.174±0.026） between the cells overexpressing ROP17 and control cells （all P<0.05）. However, the levels of Bcl-2 and Bcl-xL were not significantly different between the cells overexpressing ROP17 and the control. When the expression of c-Jun and phosphorylation of c-Jun were inhibited by c-Jun shRNA, the relative level of cleaved Caspase 3 in the RNA interferenced cells and control cells was 0.147±0.024 and 0.087±0.010, respectively（P<0.05）, and the relative level of Bcl-3 was 0.085±0.010 and 0.162±0.011, respectively（P<0.05）. Conclusion The anti-apoptosis effect of ROP17 is dependent on the phosphorylation of c-Jun and the expression of Bcl-3.

Infection of Giardia lamblia in HIV-Infected Individuals and in Kindergarden Children in Rural Area of Anhui and Genotype Analysis

YU Ying-fang, WU Xiu-ping，CHU Yan-hong, CHEN Jia-xu, TIAN Li-guang*

2016, 34(6):  11-537-541. 

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Objective To understand the situation of Giardia lamblia infection in HIV-infected individuals and in kindergarden children in rural area of Anhui Province and analyze the genotype of the parasite. Methods HIV-infected individuals registered in an AIDS treatment facility and children in a local kindergarden were included in this study during April 24 and May 9, 2015. The feces were collected, stained by iodine solution, and examined by microscopy. DNA was extracted from the positive feces, and nested PCR was performed to amplify the triosephosphate isomerase（tpi） gene  of G. lamblia. The products were sequenced. The phylogenetic tree was constructed with BLAST, ClustalX 1.83 and MEGA6.0 softwares for analysis of homology and phylogeny. Results One hundred and twenty-seven HIV-infected individuals and 125 kindergarden children were included. G. lamblia infection was found in three children and one HIV-infected individual. The infection detection rate in children and HIV patients was 2.40% （3/125） and 0.79% （1/127）, respectively （P>0.05）. Feces of the three infected children was soft, and no symptoms of diarrhea and stomachache were complained. Feces of the HIV-infected individual was washy, and symptoms like diarrhea, stomachache, weakness and weight loss were reported. PCR produced a specific band at 500 bp for the four persons. The sequencing results further confirmed infection in these four persons. The duplicate samples of the infected HIV patient had a 79% sequence similarity, and were 79% and 98% homologous to the Shanghai human strain of G. lamblia（GenBank accession No: KF271445）, respectively. The samples of the 3 children had a 99% similarity, and all were 79% homologous to the Shanghai human strain of G. lamblia. The phylogenetic tree showed that the isolate from the HIV patient was mixed genotype of A+B, while those from the 3 children were all  assemblage A. There was a high similarity between the isolates. Conclusions There is Giardia infections in HIV patients and kindergarden children in the area. The genotype of the isolate from the HIV individual is mixed assemblage A+B while those from the children are assemblage A.

Changes of Toll-like Receptor mRNA and Related Cytokines in Patients with Hepatic Alveolar Echinococcosis

ABUDUSALAMU Aini1, TUERHONGJIANG Tuxun2, MA Hai-zhang3, ZHANG Heng2, ZHANG Hao1, ABUDUKAIYOUMU Maimaiti4, LI Yu-peng2, SHADIKE Apaer2, LIN Ren-yong5, SHAO Ying-mei1, WEN Hao5*

2016, 34(6):  12-542-546. 

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Objective To investigate the expression of Toll-like receptor 2（TLR2） and TLR4 mRNA in peripheral blood mononuclear cells （PBMC） and in the liver of patients with hepatic alveolar echinococcosis （HAE）, and their correlations with related cytokines in plasma. Methods Twenty-eight HAE patients hospitalized in the First Affiliated Hospital of Xinjiang Medical University during January 2012 and June 2015 and 28 healthy volunteers as a control were enrolled in this study. Plasma levels of interferon-γ （IFN-γ）, interleukin-5 （IL-5）, IL-23, and IL-10 were measured by ELISA. qRT-PCR was performed to detect TLR2 and TLR4 mRNA levels in PBMCs and hepatic tissues. The percentage of peripheral blood eosinophil （Eo%） was determined by a hematology analyzer. The correlations of TLR2 and TLR4 mRNA levels in PBMCs with levels of related cytokines and Eo% were analyzed with the Spearman Correlation method. Results ELISA results showed that the plasma levels of IFN-γ, IL-5, IL-23, and IL-10 in the HAE group were （301.100±47.290）, （43.420±11.380）, （86.580±31.990） and （8.766±7.568） pg/ml respectively, which were higher than those in the control［（301.100±67.790）, （40.970±6.310）, （46.770±15.490） and （6.272±10.360） pg/ml］ with a statistical significance for IL-23 （P<0.01）. Results of qRT-PCR showed that the expression level of TLR2 in the HAE group （0.100±0.084） was significantly higher than that in the control （0.055±0.040） （P<0.05）, while the expression level of TLR4 in the HAE group （0.004±0.003） was comparable to that in the control（0.003±0.002）（P>0.05）. The expression of TLR2 and TLR4 mRNA in HAE lesions in the HAE group（29.680±25.650 and 21.340±16.640, respectively） were both significantly higher than that in para-lesion regions（2.308±4.140 and 5.541±9.233） and that in tissues of the control （1.112±1.431 and 1.100±1.734）（P<0.01）. There was also a significant difference in Eo% between the HAE（0.448±0.240） and the control（0.110±0.100） groups. Spearman correlation coefficients revealed a positive correlation of TLR2 mRNA in PBMCs with plasma IL-23 level and peripheral blood Eo% in HAE subjects（r=0.368, r=0.382, respectively）. Conclusion There are increases in TLR2 and TLR4 mNRA expression in PBMCs and in HAE lesions in HAE patients. The TLR2 mNRA expression in PBMCs positively correlates with plasma IL-23 level and peripheral Eo%.

An Epidemiological Survey on Echinococcosis in Yushu Prefecture of Qinghai Province

CHENG Shi-lei1, WANG Hu1, MA Xiao1, ZHANG Jing-xiao1, LIU Yu-fang1, CAI Hui-xia1, LIU Pei-yun1, MA Jun-ying1, HE Duo-long1, WU Xian-hong1, HAN Xiu-min1, WANG Yong-shun1, LIU Hai-qing1, ZHAO Yan-mei1

2016, 34(6):  13-547-551. 

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Objective To investigate the prevalence of echinococcosis in Yushu Prefecture of Qinghai Province in 2012. Methods Two to three towns were selected in each of Chengduo, Nangqian, Qu malai, Yushu, Zaduo and Zhiduo Counties from June to August in 2012. Ultrasound examination was conducted for residents aged over 1 year, and ELISA was performed to detect serum antibody against Echinococcus. Visceral dissection was performed to detect hydatid infection in rodents and livestock. ELISA was used to detect Echinococcus antigen in collected dog feces. Results A total of 7 025 residents received ultrasound examination, of whom 319 showed hydatid cysts with a morbidity rate of 4.54%. ELISA showed a serum antibody positive rate of 16.38% （457/2 790）. The mobidity of hydatid disease was highest in Chengduo County （7.41%, 181/2 444）, and the rate of serum antibody was highest in Yushu County （23.18%, 127/548）. The morbidity and serum antibody in males were 3.91% （118/3 018） and 13.93% （172/1 235） respectively, and those in females were 5.02% （201/4 007） and 18.33% （285/1 555）. In terms of age distribution, the morbidity was relatively higher in residents of 60- （8.39%, 38/453） and 40- years （6.61%, 67/1 014）; and the rate of serum antibody was highest in residents over 70 years （33.93%, 19/56）. In terms of occupation, the morbidity was relatively higher in herdsmen （5.28%, 252/4 777）, Herdsmen-peasants （6.52%, 24/368）, and religious workers（3.37%, 11/326）, while the rate of serum antibody was relatively higher in children（24%, 6/25）, religious workers （18.79%, 31/165） and herdsmen（18.34%, 328/1 788）. In terms of education level, the morbidity and the rate of serum antibody were both highest in the uneducated（5.04%, 41/4 779; 18.34%, 359/1 958, respectively）. In terms of residential pattern, the morbidity and the rate of serum antibody were both highest in those who were settled in winter and nomadic in summer （8.25%, 227/2 753; 19.48%, 158/811, respectively）. There were significant differences in the morbidity and the rate of serum antibody in aspects of residential region, sex, age, occupation, education level and residential pattern（P<0.05 or P<0.01）. In 872 rodents detected, the Echinococcus hydatid rate was 0.46% （4/872）, while in 809 cattle and sheep detected, the Echinococcus hydatid rate was 10.14% （82/809）. The fecal antigen positive rate in 838 samples of dog feces was 10.74%（90/838）. Conclusion It shows a high morbidity of hydatid diesease and serum antibody positive rate in residents, a high Echinococcus hydatid rate in cattle and sheep, and a high fecal antigen positive rate in dogs in Yushu Prefecture.

Differential Expression of Six wnt Gene Family Members in Echinococcus granulosus Protoscoleces and Adult Worms

WANG Zheng-rong1, ZHANG Yan-yan1, BO Xin-wen1*, XU Xue-ping1, XU Chun-sheng2

2016, 34(6):  14-552-557. 

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Objective To investigate the differential mRNA expression and tissue distribution of wnt ［wingless-type mouse mammary tumor virus（MMTV）integration site family，wnt］ gene members wnt1, wnt2, wnt4, wnt5,  wnt11A and wnt11B in protoscoleces and adult worms of Echinococcus granulosus. Methods The mRNA expression of wnt1, wnt2, wnt4, wnt5,  wnt11A and wnt11B was determined by qRT-PCR. Tissue distribution of wnt1, wnt2, wnt4, wnt5, wnt11A and wnt11B in Echinococcus granulosus protoscoleces was determined by the whole-mount in situ hybridization. Results The qRT-PCR results showed that the mRNA expression levels of wnt1 and wnt2 in the adult worms were 1.49 （P>0.05） and 2.53 folds（P<0.05） of those in the protoscoleces, respectively. The mRNA expression levels of wnt4, wnt5,  wnt11A and wnt11B in the protoscoleces were 25.00（P<0.01）, 33.33（P<0.01）, 14.29（P<0.01） and 1.03 folds（P>0.05） of those in the adult worms, respectively. In brief, there was no significant difference of mRNA expression in wnt2 and wnt11B between protoscoleces and adult, but there was a significant difference of mRNA expression in wnt1, wnt4, wnt5 and wnt11A between protoscoleces and adults. Results of the whole-mount in situ hybridization showed that in protoscoleces wnt1 was mainly localized in the epidermal tissue, wnt2 in suckers, wnt4 in suckers and rostellum, wnt5 and wnt11B in suckers and epidermal tissue, and wnt11A in rostellum and hooks. Conclusion The mRNA expression of wnt2 in adult E. granulosus was higher than that in protoscoleces, and the mRNA expression ofwnt4, wnt5,  wnt11A and wnt11B in protoscoleces was higher than that in the adult worms. The six wnt gene family members were all distributed in the forward region of protoscoleces.

Application of Structural Biology in Research on Toxoplasma gondii

CHEN Yan-ping1, WEI Dong-dong1, LI Hong-wei2, WEI Yan-bin1, YIN Kun1*

2016, 34(6):  15-558-564. 

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Structural biology is a rapidly-developing comprehensive discipline that interprets the atom-level assembling, interaction and movement between molecules. This paper summarizes basic methods in structural biology, with a focus on structure research progress of Toxoplasma gondii rhoptry and calmodulin-like domain protein kinase, to illustrate the application of structural biology in research on important virulence factors and drug targets of T. gondii.

Research Progress in Classification of Anopheles hyrcanus Group （Diptera ∶ Culicidae）

FANG Yuan, SHI Wen-qi, ZHANG Yi*

2016, 34(6):  16-565-570. 

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Objective The Anopheles hyrcanus group is widely distributed in the Palearctic and Oriental regions. Among the reported members, 25 species have definitive scientific names. Some are determined as vectors of malaria, lymphatic filariasis, and Japanese encephalitis. However, it is extremely difficult to morphologically discriminate the group members. With the development of techniques like crossing experiment, karyotype analysis, and molecular phylogeny, the classification of the Hyrcanus Group has been changed. In this review we update the list of the Hyrcanus Group, including addition of newly discovered species An. belenrae, An. kleini, An. xui, and An. hyrcanus spIR, and merges of An. junlianensis, An. yatsushiroensis, and An. kunmingensis as synonyms. We also discuss the relationships between An. hyrcanus and An. pseudopictus, An. lesteri and An. paraliae, as well as An. kleini and An. engarensis. This review will help to clearly define the relationships among the species.

Effect of Helminthic Infection on the Prevention and Treatment of Inflammatory Bowel Disease and the Mechanisms

TANG Chun-lian1, SHEN Zhi-qin1, LEI Jia-hui2, WANG Li-xia1*

2016, 34(6):  17-571-576. 

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Crohn’s disease and ulcerative colitis are two forms of inflammatory bowel diseases. They are autoimmune disorders caused by excessive inflammatory response to antigens in the intestine. In addition to the hygiene hypothesis which suggests the potential application of helminthic infection in the treatment of inflammatory bowel diseases, helminthic infection has shown preventive and treatment effects in animal models of inflammatory bowel disease. Clinical trials have been initiated. For example, Trichuris suis ova infection at a certain dose has a promising efficacy in the treatment of inflammatory bowel diseases. Helminthic infection may also have adverse effects. Therefore, helminth-derived immunomodulatory molecules are needed to overcome these problems. It is traditionally considered that the Th1/Th2 axis is involved in the mechnisms of the efficacy of helminthic infection. More recent research has pointed out the much more participation of the Tregs/Th17 axis. The mechanisms may also involve other palyers such as mucosal barrier, Toll like receptor and macrophages. This paper reviews the effect and mechanism of helminthic infection on the prevention and treatment of inflammatory bowel disease.

Epidemiological Analysis of Imported Malaria in Tai’an City] of Shandong Province During 2011-2015

DU Wei-ping1, ZHANG Lin-lin2, ZHANG Xin-feng1, CHEN Yong1*

2016, 34(6):  18-505-507. 

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One hundred and sixty-three imported malaria cases were reported in Tai’an City of Shandong Province during 2011-2015, consisting of 120 cases of falciparum malaria（73.6%）, 20 vivax malaria（12.3%）, 14 ovale malaria（8.6%）, 7 quartan malaria（4.3%）, and 2 unclassified cases（1.2%）. The disease onset showed no significant seasonal variation. The reported cases were more at the age of 40～49 years（70/163, 42.9%）, and were imported mainly from Africa（156/163, 95.7%）. The median interval from symptom onset to diagnosis was 4 days, and only 15 patients（9.2%） received definite diagnosis within 24 h. After medication, 161 cases recovered and 2 cases died.

Formation and Characteristics of Fasciola hepatica Metacercariae

FANG Wen，LI Tian-mei，YANG Jing，LIU Yu-hua

2016, 34(6):  19-526-528. 

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The Fasciola hepatica miracidia were used to infect 10 Galba pervia in a random manner. Beginning from day 44  after infection on which 10 metacercariae were found and a total of 495 metacercariae found in 24 days. No signs of cercaria escape or metacercaria formation in the early morning observed during 8 ∶ 00-24 ∶ 00, regardless of the environmental change. Metacercaria formation occurred mainly in the early morning. The metacercariae could attach to any object in water, but were easy to detach themselves. The findings suggest that F. hepatica cercaria escape and metacercaria formation mainly occur in the early morning in Dali.

Results of Soil-transmitted Nematode Monitoring in Henan Province in 2011-2015

DENG Yan, CHEN Wei-qi, ZHANG Ya-lan, LIN Xi-meng, ZHANG Hong-wei*, XU Bian-li

2016, 34(6):  20-533-536. 

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To understand the endemic situation of soil-transmitted nematodiasis（Ascaris lumbricoides, Trichuris trichiura and Ancylostoma sp.） in Huaiyang County, Henan Province. Over 1 000 fecal samples from inhabitants in Huaiyang County of Henan Province were collected each year during 2011-2015, in which the soil-transmitted nematodes eggs and other intestinal helminth eggs were examined by Kato-Katz technique. The helminth-positive samples were examined by filter paper culture method to identify the species of hookworm. The cellophane swab method was used to detect Enterobius vermicularis eggs in children aged 3～12 years. Soil samples were collected from vegetable field, lavatory, courtyard and kitchen of 10 families randomly selected in each year to examine Ascaris eggs by a modified saturated sodium nitrate floatation method. In 2011-2015, 5 229 people were examined and 54 person infected with intestinal helminths were found. Five intestinal helminthes, A. lumbricoides, T. trichiura, Ancylostoma duodenale, Enterobius vermicularis and Trichostrongylus orientalis were found with 13, 2, 9, 29 and 1 infection person respectively. All showed mild infection and no multiple infections were found. There was no significant difference between the year 2015 which had the highest soil-transmitted nematode infection rate 0.6%（7/1 134） and the year 2013 which had the lowest infection rate 0.3%（3/1 037）（P>0.05）. The infection rate of intestinal helminths was highest in group of <10 years（2.8%, 25/905）, followed by the groups of >70 years（1.6%, 4/256） and 30～40 years（1.2%, 8/671）（P>0.05）. The average infection rate of E. vermicularis was 1.8%（18/993） The infection rate of E. vermicularis was relatively higher in kindergarten kids（1.6%, 6/366） and students（1.3%, 13/1 005） than that in farmers（0.3%, 10/3 782）（P<0.01）. No Ascaris eggs were found in the 200 randomly collected soil samples. The intestinal helminth infection status maintaines at low level in Henan Province during 2011-2015.

Malaria Incidence and Control in Baoshan City during the Twelfth Five-Year Plan Period

LI Jia-quan, YANG He-xian

2016, 34(6):  21-577-579. 

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A retrospective analysis was made on malaria incidence in Baoshan City of Yunnan Province during the 2011-2015 Twelfth Five-Year Plan Period. The epidemiological characteristics and endemic situation of malaria were analyzed. A total of 1 301 malaria cases were reported in Baoshan City during 2011-2015, with an average incidence rate of 10.2 per 100 000 individuals, showing a relatively low prevelence of malaria. The cases were mostly imported（98.5%, 1 282/1 301）. No local malaria cases were found in Baoshan City since 2014. The cases were mostly in Tengchong City（65.6%, 853/1 301）, and mainly in the age range of 20-50 years （84.4%, 1 098/1 301）.