Table of Content

30 April 2015, Volume 33 Issue 2

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Cloning，Expression and Immunodiagnostic Evaluation of the Fasciola gigantica Thioredoxin Peroxidase

WANG Yue-qi1，ZHOU Yan1，CHENG Na1，CHEN Mu-xin1，AI Lin1，LIU Yu-hua2，

2015, 33(2):  1-81-85. 

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Objective  To immunoscreen the gene encoding thioredoxin peroxidase（TPx） from a cDNA library made from adult Fasciola gigantica worms， clone and express the gene， and evaluate the immunodiagnostic value of TPx recombinant protein.  Methods  The λ ZAP cDNA library was immunoscreened with pooled serum of fascioliasis gigantica patients. The obtained positive clones were sequenced and analyzed by multiple sequence alignment. The full-length （rFgTPx） and N-termianal truncated（rFgTPx_nt） sequence of FgTPx was subcloned into prokaryotic plasmid pET28a（+） with a non-fusion expression technique， respectively. The recombinant proteins of rFgTPx and rFgTPx_nt were purified by His-bind affinity column（Ni-NTA）. rFgTPx and rFgTPx_nt were used in indirect ELISA to test the antibody response of the serum samples. Sera of 27 fascioliasis gigantica patients， 15 patients with schistosomaisis japonica， 15 clonorchiasis sinensis patients， and 32 healthy donors were tested by using the recombinant protein based ELISA.  Results  The TPx recombinant proteins were obtained through expression， purification and renaturation， the relative molecular mass of rFgTPx and rFgTPx_nt were Mr 30 000 and Mr 26 000， respectively. The total diagnostic coincidence rate， sensitivity and specificity of rFgTPx_nt-based ELISA was 87.6%（78/89）， 66.7%（18/27）， and 96.8%（60/62）， respectively. The cross reaction with Schistosoma japonicum and Clonorchis sinensis was 0 and 1/15 for rFgTPx_nt， respectively. Before and after treatment， A450 value of the serum samples from fascioliasis patients was 0.233±0.088 and 0.129±0.072， respectively（t=4.27， P<0.01）.  Conclusion  The gene encoding TPx is expressed in the prokaryotic expression system. The recombinant protein shows proper sensitivity and high specificity for the serodiagnosis of Fasciola gigantica infection.

Screening Radius of Active Case Detection and the Malaria Parasite Rate of Carriers in China-Myanmar Border

XIAO Hui-hui1，LIU Juan2，FENG Jun1，ZHANG Shao-sen1，JIANG Wei-kang1，XIA Zhi-gui1，ZHOU Shui-sen1 *

2015, 33(2):  2-86-90. 

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Objective  To explore the effective screening radii of active case detection of the 1-3-7 surveillance and response strategy， and investigate the malaria parasite rate of carriers in China-Myanmar border.  Methods  Three villages with indigenous malaria cases in Yingjiang County of Yunan Province were selected as study sites. The persons lived around the indigenous cases（index case） within the radius of 100 m， 300 m， 500 m， and 1 km were screened by microscopy and nested PCR. Parasite rate of asymptomatic carriers at different radii were calculated.  Results  Among 278 blood samples， the parasite rate of asymptomatic carriers was 1.1%（3/278） and 2.2%（6/278） using microscopy and nested PCR， respectively. Based on the results of nested PCR， all the asymptomatic carriers could be detected within a 300 m radius around the index case， and with the highest proportion（66.7%） in the radius of 101-300 m.  Conclusion  The asymptomatic carriers of malaria parasites in the China-Myanmar border area can be effectively detected within a 300 m screening radius of index case by using nested PCR.

Establishment and Application of Multiplex PCR System for Detecting Four Human Plasmodium Species

LI Mei，XIA Zhi-gui，TANG Lin-hua*

2015, 33(2):  3-91-95. 

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Objective  To establish a multiplex PCR detection system for identifying 4 human Plasmodium species and evaluate its applicability.  Methods  The sequences of 18S rDNA gene of the 4 human Plasmodium species were compared using DNAman software， and 4 downstream primers were designed using Oligo 6.0 software， which targeted the region of variability between conserved regions 5 and 6 of the sequences. Using these primers， the specificity and sensitivity of the multiplex PCR system were evaluated， with plasmids containing the 18S rDNA gene sequence as a template. Further， a new nest PCR system(M-Nest) was established by combining the multiplex PCR system with the first-cycle genus-specific primer of the NP-1993 system. The sensitivities of the multiplex PCR system and the M-nest system were evaluated in serial dilutions of blood DNA samples from patients infected by P. falciparum and P. vivax. In addition， the NP-1993 and M-Nest systems were applied to screen the Plasmodium species in 307 blood samples from people returning to Guangxi from Ghana， a malaria epidemic area. And the NP-2002 and M-Nest systems were applied to re-check Plasmodium species in 66 blood samples collected in Guangxi from 2014 January to May， which were identified by microscopy to be infected mainly by P. ovale.  Results  The sizes of multiplex PCR products for P. falciparum， P. vivax， P. ovale， and P. malariae were 268 bp， 323 bp， 394 bp， and 446 bp， respectively， located in-between 50-bp DNA ladders. However， their melting curves had similar Tm values， thus could not be used to identify the 4 species. The minimum detection limits of P. falciparum， P. malariae， P. ovale， and P. vivax 18S rDNA gene by the multiplex PCR system were 5.58×102， 1.56×103， 1.66×103， and 1.80×102 copies/μl. The minimum detection limit of blood DNA from falciparum malaria patients by the multiplex PCR system was 1.43×102-8.84×103 copies/μl or 5.10×10-4.92×102 parasites/μl， higher than that of P. vivax（17.4-69.1 copies/μl or 13.5-83.2 parasites/μl）. Compared with this multiples PCR system, The M-Nest system further reduced the minimum detection limit of Plasmodium by 10-100 folds. Further， the M-Nest and NP-1993 systems reached inconsistent detection results in 307 blood samples from people returned to from Ghana； the former detected 2 cases of P. ovale infection while the latter failed. In addition， the NP-2002 and M-Nest systems came to the same results in re-checking Plasmodium species in the 66 blood samples.  Conclusion 　The established multiplex PCR system can identify 4 human Plasmodium species simultaneously and has good applicability in practice.

Effect of Excretory-secretory Products of Clonorchis sinensis on Nitric Oxide Production and NF-κB Activation

YANG Qing-li1，2，JIANG Zhi-hua2，SHEN Ji-qing3，CHEN Ying-dan1，ZHOU Xiao-nong1 *

2015, 33(2):  4-96-100. 

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Objective  To study the role of excretory-secretory products（ESPs） from Clonorchis sinensis in the production of nitric oxide （NO） and the activation of nuclear transcription factor kappa B（NF-κB） in the macrophages of RAW264.7 mouse.  Methods  20 μg/ml of C. sinensis EPSs， the organic solvent extracts of EPSs（ESP-ex）， and 0.1 μg/ml of lipopolysaccharide from Salmonella minnesota（LPS-SM） were used as stimulators in co-culture with RAW264.7 mouse macrophages as experimental groups. The Hank’s balanced salt solution（HBSS） served as control. At the same time the RAW264.7 macrophages were stimulated with EPSs， ESP-ex， and LPS-SM， and then added 0.3 mmol/L of SMT， a specific inhibitor of iNOS as the interference groups. After co-culture for 18 days， the concentrations of NO2- in the culture supernatants were detected with Griess regents， and the activation of NF-κB was determined by transfection with a NF-κB-inducible reporter plasmid， pNiFty2-SEAP. The activities of secreted embryonic alkaline phosphatase（SEAP） in culture supernatants were quantified by using HEK-BlueTM detection medium and expressed as the value of optical density at 620 nm（A620 value）. The intercellular activities of SEAP were determined by microscopic observation.  Results  After stimulation with both ESPs-ex and LPS-SM， the concentrations of NO2- in culture supernatants were（14.30±1.62） and （14.10±2.17） μmol/L， respectively， which were significantly higher than that of the control［（7.70±0.95） μmol/L］（P<0.05）， and significantly decreased to （8.97±0.81） and （4.96±1.36） μmol/L after adding SMT， respectively（P<0.05）. However， the concentration of NO2- in ESPs stimulation group ［（4.06±0.62） μmol/L］ was lower than that of the control（P<0.05）， and almost unchanged［（3.99±0.87） μmol/L］ after adding SMT（P>0.05）. SEAP activity in ESP group（0.836±0.005） was significantly higher than that of the control ［（0.097±0.009） μmol/L］（P<0.05）. A strong blue color reaction was observed in cells of ESP group. SEAP activity of ESPs-ex and LPS groups［（0.112±0.004）， （0.116±0.009） μmol/L］ was slightly higher than that of the control（P>0.05）， and blue color reaction was observed in some cells.  Conclusion  ESPs from C. sinensis can stimulate NF-κB activation in RAW264.7 cells. The water-soluble components of ESPs can inhibit the NO production， while ESPs-ex and LPS-SM can promote the NO production.

Plasma Metabolism and Protective Effect of Oral Administration of Niclosamide on Schistosoma japonicum Cercarial Invasion in Mice

TU Zhen，JIANG Bin，XUE Jian，TAO Yi，WEI Yu-fen，ZHANG Hao-bing*

2015, 33(2):  5-101-104. 

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Objective  To study the metabolism of niclosamide in plasma， and the protective effect of its oral administration on Schistosoma japonicum cercarial invasion in mice.  Methods  Twenty-four female Kunming mice were randomly divided into 8 groups， each with 3 mice. Each mouse was treated orally with 120 mg niclosamide per kilogram of body weight （120 mg/kg）. The plasma samples were collected at 0.25， 0.5， 1， 2， 4， 8， 16， and 24 h after treatment by retro-orbital blood sampling. The blood drug concentration was determined by HPLC. The pharmacokinetics parameters were calculated such as peak concentration（Cmax）， peak time（Tmax）， mean residence time（MRT）， and elimination half life（T1/2）. Thirty Kunming mice were randomly divided into 6 groups. Among them， 5 groups were treated orally with 40， 80， 120， 160， and 200 mg/kg niclosamide， respectively. The remaining untreated group served as control. One hour post-treatment， each mouse was infected with 40±2 Schistosoma japonicum cercariae. Another 35 mice treated with 200 mg/kg niclosamide were randomly divided into 7 groups. Mice in each group were infected with 40±2 S. japonicum cercariae on 0.25， 1， 4， 8， 12， and 24 h after treatment， named as group A， B， C， D， E， and F. Five untreated mice served as control（group G）. All mice were sacrificed 35 days post-infection. Mean worm burden and worm reduction were calculated.  Results  At a dose of 120 mg/kg niclosamide， the blood drug concentration was （0.40±0.28） μg/ml at 0.25 h post-treatment， reached a peak of （0.91±0.34） μg/ml at 1 h， and decreased to （0.49±0.38） μg/ml at 2 h， and got close to 0 at 16 h. The mean residence time（MRT） in mice was （6.78±1.47） h， and the elimination half time was （6.80±7.05） h. No significant difference was found in worm burden between different dose groups and control group（P＞0.05）. The mean worm burden in group A was significantly lower than that of the control （P＜0.05） with a mean worm reduction of 79.1%. And there was no significant difference in worm burden between other groups and the control（P＞0.05）.  Conclusions  The blood drug concentration increases rapidly by gavage administration of 120 mg/kg niclosamide， reaching to the maximum concentration at 1 h post-treatment. It shows a certain potective effect of oral administration of 200 mg/kg niclosamide on Schistosoma japonicum cercarial invasion at 0.25 h after treatment.

A Duplex PCR Method for Detection of Babesia caballi and Theileria equi

ZHANG Yang 1，ZHANG Yu-ting 1，WANG Zhen-bao 2，BOLATI 3，LI Hai 3，BAYINCHAHAN 1 *

2015, 33(2):  6-105-109. 

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Objective  To develop a duplex PCR assay for detection of Babesia caballi and Theileria equi.  Methods  Two pairs of primers were designed according to the BC48 gene of B. caballi and 18 s rRNA gene of T. equi， and a duplex PCR assay was developed by the optimization of reaction conditions. The specificity， sensitivity and reliability of the method were tested. The horse blood samples of suspected cases were collected from Yili region， and detected by the duplex PCR， microspopy， conventional PCR， and fluorescence quantitative PCR， and the results were compared.  Results  Using the duplex PCR assay， the specific fragments of 155 bp and 280 bp were amplified from DNA samples of B. caballi and T. equi， respectively. No specific fragment was amplified from DNA samples of B. bigemina、 Theilerdia annulata， Theilerdia sergenti， Toxoplasma gondii， Neospora caninum， and Trypanosoma evansi. The limit of detection was 4.85×105 copies/μl for B. caballi DNA and 4.85×104 copies/μl for T. equi DNA， respectively. Among the 24 blood samples， 11 were found B. caballi-positive by the duplex PCR assay， and 18 were T. equi-positive. The coincidence rate of microscopy， conventional PCR， and fluorescence quantitative PCR with duplex PCR was 91.7%（22/24）， 95.8%（23/24）， and 95.8%（23/24）， respectively.  Conclusion  A duplex PCR assay for simultaneous detection of B. caballi and T. equi is established.

The Levels of IL-4，IL-9，and IgE in Patients Infected with Intestinal Helminths and their Clinical Values

GUO Ai-ye1 *，LIN Xi-meng2，ZHANG Yu-qin2，WU Hui3

2015, 33(2):  7-110-104. 

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Objective  To investigate the serum levels of interleukin-4（IL-4）， interleukin-9（IL-9）， and immunoglobulin E（IgE） in the patients infected with intestinal helminths， and study their relationship to the clinical symptoms or species of the helminths.  Methods  This study was carried out in the Department of Paediatrics， Henan Provincial People′s Hospital from January 2010 to July 2014. The blood samples were collected from 55 infected patients. Among the 55 cases， 18 cases（32.7%） were with ascaris infection， 8 cases（14.5%） of hookworm infection， 7 cases（12.7%） of whipworm infection， and 22 cases（40%） of pinworm infection. ELISA were used to measure the levels of IL-4， IL-9， and IgE in peripheral blood samples from the patients and 15 healthy volunteers. The relationship between the concentration of the cytokines and clinical symptoms or species of the parasites was analyzed.  Results  The serum levels of IL-4， IL-9， and IgE in infection group were （157.42±41）pg/ml， （59.9±21.7）pg/ml， and （316.6±129）IU/ml， respectively， which were higher than that of the healthy control[ IL-4（39.01±23.5）pg/ml， IL-9 （21.3±12.5）pg/ml， IgE （127.7±57.6）IU/ml]（P<0.01）. After treatment by albendazole in the infection group, the level of IL-4， IL-9， and IgE decreased to （98.1±41.7）pg/ml， （38.7±14.1）pg/ml， and （253.1±94.0）IU/ml， respectively, but still higher than that of the control（P<0.05）. IL-9 level in patients with upper gastrointestinal hemorrhage was （76.1±23.5） pg/ml， which was higher than that of those with abdominal discomfort or disruption to bowel habits[（54.3±22.1）pg/ml]（P<0.05）， but lower than that of those with allergic dermatitis [（108.5±33.4）pg/ml] （P<0.05）. No significant difference was found in the levels of IL-4 and IgE among the above three groups. The level of IL-9 in patients infected with pinworms was（120.3±41.0）pg/ml， which was higher than that of ascaris infection group[（90.1±29.7）pg/ml]， hookworm infection group[（77.3±18.3）pg/ml]， and whipworm infection group[（62.5±24.3）pg/ml]（P<0.01）. There was no significant difference in the serum level of IL-9 between ascaris infection group and hookworm infection group（P＞0.05）， whereas the IL-9 level in ascaris infection group and hookworm infection group was higher than that of whipworm infection group[（62.5±24.3）pg/ml]（P<0.01）. There were no significant difference in the serum level of IL-4 and IgE among the patients infected with the species of different helminthes（P＞0.05）.  Conclusion  The levels of IL-4， IgE， and IL-9 are considerably related with intestinal helminth infection， while IL-9 level varied with different helminth species and clinical symptoms.

Effect of Echinococcus multilocularis Cyst Fluid on the Expression of Five MAPK-pathway Genes of Rat Hepatic Stellate Cells

REN Bin，FAN Hai-ning*，DENG Yong，WANG Hai-jiu，REN Li

2015, 33(2):  8-114-117,121. 

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Objective  To investigate the effect of Echinococcus multilocularis cyst fluid on five MAPK （mitogen-activated protein kinase）-pathway genes of rat hepatic stellate cell.  Methods  Rat hepatic stellate cell line， HSC-T6 cells were co-cultured with different protein concentrations of E. multilocularis cyst fluid（0.01， 0.025， 0.05， 0.1， 0.2， 0.4， 0.9， 1.7， 3.4， 6.8， and 13.5 mg/ml） for 24 h. HSC-T6 cells cultured with complete medium served as control group. The morphological change of cells was observed under the microscope. The expression of extracellular signal-regulated kinase （ERK）， c-Jun N-terminal kinase （JNK） and mitogen-activated protein kinase（p38） in HSC-T6 cells was detected by real time fluorescent quantitative PCR.  Results  After co-cultured for 24 h， most HSC-T6 cells in 13.5 mg/ml group shrank as a precursor to slough off； In 6.8 mg/ml group， some HSC?鄄T6 cells shrank and changed to long fusiform shape with many slender pseudopodia； In 3.4 mg/ml group， most HSC-T6 cells showed as adherent cells with an irregular polygon shape， formed a sheet with short pseudopodia. There was no difference in cell morphology between <1.7 mg/ml groups and control group. When the protein concentration was above 1.7 mg/ml， the mRNA level of ERK1/2， JNK1/2， and P38 increased significantly increased. In 6.8 mg/ml cyst fluid group， the mRNA level of ERK1/2， JNK1/2， and P38 was higher than that of the control （P<0.05）.  Conclusion  6.8 mg/ml Echinococcus multilocularis cyst fluid can have a significant impact on mRNA levels of ERK1/2， JNK1/2 and p38 in rat hepatic stellate cells.

Study on the Resistance of Culex tritaeniorhynchus to DDT and Deltamethrin in Yunnan Province

JIANG Jin-yong1，2，ZHOU Hong-ning2，ZHENG Yu-ting2，WANG Jian2，YANG Rui2，MA Ya-jun1 *

2015, 33(2):  9-118-121. 

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Objective  To investigate the resistance level of Culex tritaeniorhynchus to DDT and deltamethrin in Yunnan Province.  Methods  Adult Culex tritaeniorhynchus samples were collected in Zhaoyang District of Zhaotong City， Mangshi County of Dehong Prefecture， Yuanjiang County of Yuxi City， Jiangcheng and Menglian County of Puer City. The susceptibility of Cx. tritaeniorhynchus to DDT and deltamethrin were tested by bioassay method. The resistance level was judged by adjusted mortality.  Results  Culex tritaeniorhynchus collected from Zhaoyang, Mangshi, Jiangcheng, Menglian, and Yuanjiang, and exposed to DDT for 1 h， the mortality after 24 hours was 51.1%, 86.8%, 35.4%, 21.0%, and 4.6%， respectively; the resistant grade in Mangshi was maybe resistance（M）， and the others 4 sites were resistance（R）. The range of KT50 to DDT was from 18.76 min to 395.65 min. The mortality of the mosquitoes from the five sites to deltamethrin was 36.9%, 59.2%, 43.1%, 34.1%, and 3.3%， respectively; the resistant grade was R in all sites， and the range of KT50 was 8.69-715.37 min.  Conclusion  Culex tritaeniorhynchus in Yunnan Province shows simultaneously resistant to DDT and deltamethrin, and therefore the insecticiding strategy should be adjusted.

Assessment of Serum IgG and Its Subclasses in Cystic Echinococcosis Patients and Its Application for Diagnosis

ZHANG Ting1，2，CHEN Ying1，ZHANG Jun-rui3，WANG Dong4，E Zheng-long3，FENG Yu4，MO Xiao-jin1，SUN De-jian1，XU Bin1*，HU Wei1

2015, 33(2):  10. 

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Objective  To assess the diagnostic performance of hydatid cyst fluid（HCF） in detecting the anti-HCF IgG and its subclasses IgG1， IgG2 and IgG4 in serum of cystic echinococcosis patients.  Methods  ELISA was used to measure IgG and its subclasses IgG1， IgG2， and IgG4 specific for Echinococcus granulosus HCF， in the sera of 37 cystic echinococcosis（CE） patients and 29 healthy subjects in Huan County， Gansu Province. The receiver operating characteristic（ROC） curves of the four antibodies were analyzed by MedCalc software， referenced with the gold-standard B ultrasonic imaging. The diagnostic performances between the four antibodies were compared using paired z statistics based on the areas under the curve（AUC）， and the best diagnostic threshold was determined for each. The sensitivity and specificity for detecting the four types of antibodies were compared using Chi-square test.  Results  The AUCs for IgG and its subclasses IgG1， IgG2， and IgG4 were 0.722， 0.919， 0.712， and 0.835， respectively； the AUC of IgG1 was significantly higher than those of IgG（z=3.629， P<0.05） and IgG2（z=3.292， P<0.05）. The sensitivity for detecting IgG， IgG1， IgG2， and IgG4 was 54.1%， 91.9%， 67.6%， and 75.7%， respectively； the sensitivity for IgG1 was significantly higher than that for IgG（χ2=3.84， P<0.05）， IgG2（χ2=6.80， P<0.05）， and IgG4（χ2=10.16， P<0.05）. The specificity for the four antibodies was 89.7%， 82.8%， 72.4%， and 89.7%， respectively， and no significant difference was found between them. In addition， the sensitivity for detecting IgG4 antibody was significantly higher in CEⅠ-Ⅲ than in CEⅣ-Ⅴ patients.  Conclusion  The IgG1 antibody shows the highest detection sensitivity by HCF， thus having potential value in diagnosis of cystic echinococcosis.

Diagnosis and Treatment for Four Cases of Sparganosis mansoni

SHEN Mei-qing*，LIU Jian，YANG Li-qun，ZHANG Hong-fang，QIAN Hua

2015, 33(2):  11-125-127. 

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The data of 4 sparganosis mansoni cases were collected from January 2010 to September 2014， and analyzed by descriptive epidemiological methods. Among the cases， 3 cases had a history of eating raw frogs， and 1 case had a history of eating half-cooked frogs and drinking unboiled water. All cased and 3 out of 7 persons eating raw frogs together with case 3 were positive for anti-Sparganum mansoni antibody. 2 patients were cured by operation removal and praziquantel+albendazole treatment， and the other 2 cases were cured by drugs only.

Percentage of Th17 Cells in Spleen and IL-17 Level in Bronchoalveolar Lavage Fluid in Dermatophagoides farinae Allergic Asthma Mice

LV Qin1，YANG Xiao-meng2，XIAO Xiao-jun2，CHEN Si2，YANG Ping-chang2，LIU Zhi-gang2，ZHANG Min1*

2015, 33(2):  12-127-129. 

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Objective  To detect the percentage of Th17 cells in spleen and IL-17 level in bronchoalveolar lavage fluid in Dermatophagoides farinae allergic asthma mice.  Methods  Twenty BALB/c mice were randomly divided into control group（n=10） and asthma group（n=10）. Mice in control group were treated with PBS plus 2 mg Al（OH）3 and those in asthma group were sensitized with 200 μl solution［50 μg Dermatophagoides farinae crude extracts plus 2 mg Al（OH）3］ on day 0， 7 and 14. One week after the last sensitization， all mice were intranasally challenged with 50 μg Dermatophagoides farinae crude extracts daily for 7 days. Twenty-four hours after the last challenge， mice were sacrificed. The sera， bronchoalveolar lavage fluid（BALF） and spleens were collected. The serum levels of IgE and IgG1， and IL-17 level in BALF were determined by ELISA. The percentage of Th17 cells in spleen was tested by flow cytometry.  Results  The serum levels of IgG1 and IgE in asthma group were（0.10±0.01） pg/ml and （1.15±0.10） pg/ml， respectively， which were higher than that of the control［（0.06±0.01） pg/ml and （0.04±0.01） pg/ml］（P<0.05）. IL-17 level in asthma group（85.13±2.36） pg/ml was higher than that of the control[（48.27±4.14） pg/ml]（P<0.01）. The percentage of Th17 cells in asthma group[（5.19±0.68）%] was also higher than that of the control[（0.95±0.19）%]（P<0.01）. Meanwhile， the percentage of Th17 cells in spleen was positively correlated with IL-17 level in BALF（r=0.851， P<0.01）.  Conclusion  Compared with healthy mice， both the percentage of Th17 cells in spleen and IL-17 level in BALF have increased significantly in Dermatophagoides farinae allergic asthma mice.

Scanning Electron Microscopic Observation on Adult Gnathostoma doloresi Worms and the Phylogenetic Analysis of G. doloresi Based on ITS2 and COX1 Gene Sequences

LI Wen-wen1，2，Ren Yi-jing2，LI Jian3，HUANG Wei-yi2，4 *

2015, 33(2):  13-130-134. 

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Objective  To observe the ultrastructure of adult Gnathostoma doloresi worms isolated from wild boar by using scanning electron microscope（SEM）， and analyze its phylogenetic relationships based on ITS2 and COX1 gene sequences.  Methods  Two adult G. doloresi worms were fixed by glutaraldehyde and osmium peroxide. Ultrastructural characters of those samples were observed under SEM. Amplification and sequencing of the ITS2 and COX1 genes were performed following the extraction of total genomic DNA. Sequence analysis was performed based on multiple alignments and phylogenetic analysis was made by Neighbor-Joining method using MEGA 6.0.  Results  The bottle-shaped adult worm covered with numerous small spines. The cervical groove connected head bulb and body without spines. There was obvious distinction in body spines which surround cervical papillae and swollen area in the middle part of the body. The fragments of ITS2（418 bp） and COX1（381 bp） gene were obtained by PCR combined with sequencing， and were registered to the GenBank database with the accession No. of JN408329 and JN408299， respectively. The BLAST results showed that， two sequences had 99% and 98% consistency with G. doloresi ITS2 （GenBank accession No. AB181156） and COX1 （No. AB180100） gene sequences, respectively. The phylogenetic tree indicated that the two G. doloresi worms were at the same clade with a bootstrap value at 100% and 85% based on the ITS2 and COX1 sequences， respectively. G. doloresi and G. hispidum were also clustered together.  Conclusion  The results provide adequate information for the SEM morphological data of adult G. doloresi worms， and its phylogenetic relationship.

Application of Nano Carbon-based Immunosensor in Pathogen Detection

CHEN Xiao-heng1，WANG Miao2，LU Shao-hong1 *

2015, 33(2):  14-135-141. 

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The biosensors exhibit many advantages such as simple operation， rapid reaction and high sensitivity in pathogen detection. The sensitivity and specificity of the biosensors can be significantly enhanced by the combined use of carbon nano-materials（such as carbon nanotubes and graphene） and bio-sensing devices. This paper reviews the characteristics of carbon nano-biosensors， its applications in pathogen detection and new development.

Progress on the Relationship between Clonorchis sinensis Infection and Cholangiocarcinoma

WANG Cai-qin，YU Xin-bing，LI Xue-rong*

2015, 33(2):  15-142-146. 

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Currently， 12.49 million people are infected with Clonorchis sinensis in China. The incidence of bile duct carcinoma increased for recent years. More than a century ago， some scholars have put forward the idea about the relations between C. sinensis infection and cholangiocarcinoma， and committed to research the mechanism. However， the intrinsic mechanisms involved in these processes remain obscure. It is therefore important to pay more attention to the further investigation of the relevance between C. sinensis infection and bile duct carcinoma. This review summarizes the possible mechanism of cholangiocarcinoma caused by C. sinensis， which is displayed on mechanical damage， stimulation of the worms and their excretory-secretory products（ESP）， abnormity of immunoreaction and molecular genetic lesions.

The Role of Dendritic Cells in Host Immunity against Helminth Infections

JIANG Jing1，2，ZHAO Quan2，YANG Gui-lian1 *

2015, 33(2):  16-147-150. 

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 Dendritic cells are the strongest professional antigen-presenting cells. Helminth infection could promote maturation of immature dendritic cells， induce Th2-type immune responses， and inhibit the normal function of dendritic cells which is closely associated with the immune evasion. This paper reviews the role of dendritic cells in host immunity against helminth infections.

Analysis on Research Projects Supported by the National Natural Science Foundation of China at the National Institute of Parasitic Diseases during 2003-2013

ZHOU Xiao-jun，ZHENG Bin*，YI Feng-yun，XIONG Yan-hong，ZHANG Min-qi

2015, 33(2):  17-151-153. 

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The data of the National Natural Science Foundation（NSFC） projests obtained by the National Institute of Parasitic Diseases （NIPD）， Chinese Center for Disease Control and Prevention （China CDC） during 2003-2013 were collected from internet-based science information system of NSFC， and NSFC search tool of Dingxiang Garden （http：//nsfc.biomart.cn/）. The number of funded projects， their subject classification and approved amount were analyzed， and compared with the other institutes of China CDC. Furthermore， the rationalization proposals were given in order to enhance the level of foundation management in the future．

Toxoplasma gondi Antibody Profile in Patients with Leukemia or Lymphoma

TIAN Meng-yuan1，HUANG Yun-hong2，HU Yun-fei2，PENG Feng-tao1，ZOU Cai-yan1，LI Yong-nian1 *

2015, 33(2):  18-151,153-155. 

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Blood samples were collected from patients with leukemia（n=150） or lymphoma（n=150） in the Cancer Hospital from March to September 2014. The specific antibodies（IgG， IgM）to， and circulating antigens（CAg） of Toxoplasma gondii were determined by ELISA. A 529 bp specific sequence was amplified by PCR from the genomic DNA of T. gondii. T. gondii-specific IgG positive rate in patients with leukemia and lymphoma were 16.0%（24/150） and 20.0%（30/150）， respectively， which were significantly higher than that of healthy persons（6.4%， 7/110）（P＜0.05）. IgM positive rate of the leukemia patients， lymphoma patients， and healthy persons was 2.7%（4/150）， 1.3%（2/150）， and 0.9%（1/110）（P＞0.05）， respectively. No significant difference was found in IgM and CAg positive rate among leukemia patients， lymphoma patients， and healthy persons （P＞0.05）. No specific band （529 bp） was detected in all samples.

Tracing Investigation of One Vivax Malaria Case by Detecting the Gene Encoding Circumsporozoite Protein in Henan

LIU Ying，QIAN Dan，CHEN Wei-qi，ZHOU Rui-min，YANG Cheng-yun，

2015, 33(2):  19-156-178. 

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A vivax malaria case in Henan Province was diagnosed as an indigenous case firstly in June 2013， and replased in April 2014. The clinical data of this case were collected and the epidemiological investigation was conducted. The blood samples were examined by Giemsa-stained blood smear， rapid diagnostic test strip（RDT） and nested PCR. This patient stayed at Myanmar for about one week in May 2013， had the symptoms of chills， fever and sweating in June， and was diagnosed as vivax malaria. After treated with artesunate， the symptoms disappeared. The CSP sequence was amplified from the blood samples of the first and second attack， and there was no difference in the central repeat domain of CSP gene. The identity of our two CSP gene sequences to that of Myanmar isolates（GenBank accessssion No. ABS95455， ABS95456） was 95.1% and 100%， while their nucleotide sequence was with 88.8% and 67.1% identity with that of Henan isolates （accessssion No. KP888996, KP889000）， respectively. This patient is therefore confirmed as an imported relapse case of Plasmodium vivax infection.

Expression，Purification and Bioinformatics Analysis of β-hexosaminidase of Dermatophagoides farinae

MO Li-hua1，LIU Yu-lin2，YANG Li-tao3，FAN Xiao-qin3，LIU Zhi-gang1，YANG Ping-chang 1 *

2015, 33(2):  20-159-161. 

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The DNA fragment encoding β-hexosaminidase was synthesized， and cloned into pET-28a vector. The constructed plasmid pMD18-T-β-hexosaminidase was transformed into E. coli Top10 and followed by expression of the protein induced by IPTG. SDS-PAGE result showed that the relative molecular mass of the recombinant protein was about M_r 55 000. The full length of β-hexosaminidase gene was 1 410 bp. Bioinformatics analysis revealed that β-hexosaminidase was composed with 469 amino acid residues with a calculated molecular weight of M_r 55 000， and its secondary structure was composed of strand（14.71%）， helix（30.70%）， and loop（54.58%）. β-hexosaminidase was a hydrophilic protein without signal peptide， and located in the extracellular space.