Table of Content

30 June 2014, Volume 32 Issue 3

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Cloning and Characterization of Conservative Region of Tyrosine Kinase 4, A Novel Gender-Associated Gene of Schistosoma japonicum

Zang-Wei, LU Wei-Yuan, XU Yu-Xin, CAO Jian-Ping

2014, 32(3):  1-167-171. 

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Objective  To clone and express the conservative region of gene encoding tyrosine kinase 4 of Schistosoma japonicum and identify the difference in gene expression between genders of S. japonicum.  Methods  The gene fragment was amplified by reverse transcriptase-polymerase chain reaction（RT-PCR）using the total RNA isolated from adult S. japonicum（Chinese strain）with primers designed according to SmTK4 encoding tyrosine kinase 4. The purified PCR product was ligated with pET28a and the recombinant protein was induced to express, and analyzed by SDS-PAGE, Western blotting and tools of bio-informatics. Subsquently, total RNA was respectively isolated from adult males, females and both worms of S. japonicum. The real-time PCR was performed with corresponding primers after reverse transcription to show the expression levels of the gene in both genders.  Results  A 582 bp in size of the DNA fragment was acquired by RT-PCR. Sequence analysis indicated that the fragment showed 91% in homology to that of SmTK4, and the deduced amino acid sequence showed to be 98% identical with that encoded by SmTK4. SDS-PAGE analysis revealed that the relative molecular weight（M_r）of expressed protein rSjTK4 was approximately 26 000. The bio-information analysis demonstrated that the protein had multiple sites of enzymatic activities. The relative number of copies of SjTK4 in male worms was 0.61±0.29, while 0.03±0.02 in female worms, showing that the mRNA level of TK4 in male worms was 18 times higher than that in females.  Conclusion  The conservative region of gene encoding tyrosine kinase 4 of S. japonicum is successfully cloned and expressed. The mRNA level of TK4 in male worms is significantly higher than that in females.

Effect of Toll-like Receptor（TLR）7 Deficiencies on the In Vivo Immune Response against Schistosoma japonicum

JIANG Yan-Yan, XU Yu-Xin, YUAN Zhong-Ying, SHEN Yu-Juan, WU Ying, LIU Hai-Peng, HU Yuan, CAO Jian-Ping

2014, 32(3):  2-172-175. 

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Objective  To explore the toll-like receptor 7 knocked out（TLR7^-/-） mice immune response against Schistosoma japonicum.  Methods  C57BL/6 mice（WT） and TLR7^-/- mice（TLR7^-/-） were infected with 20 S. japonicum cercariae via shaved abdomen. There were nine mice in each group. At 6 weeks post-infection, mice were sacrificed. Adult worms were harvested by perfusion of the portal venous system, and the number of adult worms was determined. At the time of perfusion, livers were collected, weighed, and digested overnight with 5% potassium hydroxide, and eggs were counted. In addition, spleens were aseptically harvested when WT and TLR7^-/- mice were sacrificed at day zero and 6 weeks after S. japonicum infection. After 72 hours of the co-culture with or without S. japonicum eggs, the culture supernatants were collected for cytokine assays by ELISA assay.  Results  At 6 weeks after infection, there was no significant difference in number of worms [(10.5±3.3) vs (9.8±5.2)] and eggs per gram of liver tissue [(38 251.9±4 891.5) vs (38 160.9±3 341.0)] between WT and TLR7^-/- mice. As for Th1/Th2 cytokine secretion from spleen cells, the levels of TNF-α［（43.7±9.8） pg/ml］ and INF-γ［（215.2±35.4） pg/ml］ from TLR7-/- infected mice were lower than those of WT infected mice［（63.4±22.9） pg/ml, （383.5±253.3） pg/ml］. For Th2 cytokines detection, the production of IL-10 ［（1 702.6±572.3） pg/ml］ and IL-4 ［（59.5±10.1） pg/ml］ from TLR7^-/- mice were higher than those of WT mice ［（595.2±386.3）pg/ml，（8.3±0.9）pg/ml］（P<0.05, P<0.01）, while IL-4 level ［（63.9±33.9） pg/ml］ from TLR7^-/- infected mice was higher than those of WT infected mice［（23.3±11.5） pg/ml］.  Conclusion  TLR7-/- mice has a dominant Th2 response under the normal state. The absence of TLR7 does not influence the immune response against S. japonicum infection at 6 weeks post-infection.

In Vitro Effect of Photoactivated Hypericin on Anti-Schistosoma japonicum Adult Male Worms

CAI Ru, SHE Xin-Ping, WANG Yu, GONG Wei, ZHANG Hui-Qin, XIA Chao-Ming

2014, 32(3):  3-176-179. 

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Objective  To investigate the in vitro effect of photoactivated hypericin on anti-Schistosoma japonicum adult male worms.  Methods  Kunming mice were infected with 60-80 Schistosoma japonicum single-sex cercariae. At 6 weeks post-infection, the mice were sacrificed and adult male worms of S. japonicum were collected. The worms were incubated in DMEM medium containing different concentrations of hypericin （0.1, 0.2, 0.5, 1.0, 1.5, 2.0, and 2.5 μmol/L） in the presence or absence of light. In photoactivated hypericin groups, after 6 h of dark incubation the worms were exposed to LED light irradiation（590 nm） for 30, 60, 90, and 120 min, respectively, and then cultured overnight in darkness（16 h）. In the next morning, the parasites were washed, resuspended in drug-free medium, and incubated in the dark for 48 h. These worms were observed with stereomicroscopy and scanning electron microscopy（SEM）.  Results  Photoactivated hypericin showed the ability to kill Schistosoma japonicum in vitro. The death rate was 20% in 0.1 μmol/L photoactivated hypericin group under 30 min irradiation, and 100% in 2 μmol/L under 90 min irradiation and 2.5 μmol/L under 60 min irradiation, respectively. In blank control group, DMSO control group, and hypericin groups without light irradiation, worms were alive. After 60 min irradiation, the worms in 1.0, 2.5, 5.0 μmol/L photoactivated hypericin groups showed spastic paralysis characterized by reduced body length, pronounced tight curl, body stiffness, and complete cessation of movement. Surface tegumental damages of adult worms in 2.0 μmol/L photoactivated hypericin group for 60 min irradiation were observed under SEM, such as vacuole formation, erosion and peeling of the tegument, collapse of the sensory papillae, and even the normal structure disappeared completely. Both death rate and morphological damage of the worms treated by photoactivated hypericin were positively correlated with hypericin dose and light irradiation time.  Conclusion  Photoactivated hypericin has anti-Schistosoma japonicum adult male worms effect in vitro.

Assessment on the Effect of Joint Effort for Schistosomiasis Control in Hubei Province

XU Jing, ZHANG Xian-Feng, GAO Jing, HUANG Xi-Bao, ZHANG Li-Juan, LI Shi-Zhu, CAO Chun-Li, CHU Hong-Qing, YU Qing, DANG Hui, BAO Zi-Ping, JIA Tie-Wu, CHEN Zhao, WANG Li-Ying, ZHOU Xiao-Nong, HAO Yang

2014, 32(3):  4-180-185. 

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 Objective  To analyze the progress of implementation of integrated strategy with emphasis on the control of infectious sources and effectiveness for joint-project of schistosomiasis control in Hubei province.  Methods  Data on the endemic status and implementation of each integrated intervention in 6 collaborated counties including Gongan, Hanchuan, Honghu, Jiangling, Xiantao and Yangxin during 2009-2013 were collected and analyzed. 18 administrative villages with a history of endemic schistosomisis from 6 counties were selected for field survey. Individuals aged 6-65 years received screening test by IHA, and feces of antibody positive inhabitants were collected and tested by miracidia hatching technique. Hatching technique was conducted to determine the infection rate of schistosomiasis in cattle if there was any cattle existed.  Results  Various interventions were conducted with adaption to the local situation by the Departments of Agriculture, Water Conservancy, Forestry, and Health. The total number of cattle decreased from 75 388 at the beginning of 2009 to 1 805 at the end of 2013 in 6 counties with a reduction rate of 97.5%, while the prevalence in cattle reduced to 0-0.3% in 2013. Snail-infested areas were stable but areas with infected snails decreased significantly, and no infected snails were found in 2012-2013. Meanwhile, the infection rate of human beings on county level were less than 1%. No infected snails and cattle were found in 18 selected villages and the prevalence in inhabitants was in the range of 0-0.8%.  Conclusion  Cooperation between provincial government and the Ministries of Health and Agruiculture accelerates the process to reach the criteria of transmission control of schistosomiasis in Hubei Province. However, sustainable effort in needed as the current endemic situation of schistosomiasis is stillunstable.

Efficacy of Albendazole Chitosan Microspheres against Echinococcus granulosus Infection in Mice

LIANG Wen, WANG Xin-Chun, WU Xiang-Wei, ZHANG Shi-Jie, SUN Hong, MA Xin, PENG Xin-Yu

2014, 32(3):  5-188-192. 

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Objective  To observe the therapeutic effect of albendazole chitosan microspheres （ABZ-CS-MPs） on cystic echinococcosis in mice.  Methods  Two hundred male kunming mice were each infected by intraperitoneal inoculation of about 5 000 viable protoscoleces of Echinococcus granulosus. Another 20 mice were kept as blank control. After 12 weeks post infection, the mice were randomly divided into four groups named as infection control group（n=20）, ABZ-CS-MPs group, albendazole liposome（L-ABZ） group, and albendazole tablet group. The latter three treatment groups were then each divided into three subgroups （n=20） by given the dose of 37.5, 75.0, and 150.0 mg/kg for three times per week, respectively. After 12 weeks of treatment, all mice were sacrificed. The weight of hydatid cysts was measured and the inhibition rate were calculated. Mouse liver was observed. The histopathological changes of E. granulosus were observed by microscopy. The concentration of albendazole sulfoxide in plasma and liver tissues was determined by high-performance liquid chromatography.  Results  Compared with the other treatment groups, the turbidity of contained fluid, the consolidation level and calcification level of hydatid cysts in ABZ-CS-MPs group were higher. The average weight of hydatid cysts in each treatment group was lower than that of infection control group［（3.19±2.94） g］（P<0.05）. The cyst weight in 37.5, 75.0, and 150.0 mg/kg ABZ-CS-MPs group［（0.28±0.28）, （0.24±0.22）, and （0.20±0.19） g, respectively］ was lower than that of albendazole tablet groups ［（0.77±0.74）, （0.55±0.42）, （0.76±0.35） g］（P<0.05）. Among the same dosage groups, the inhibition rate in ABZ-CS-MPs group（from low to high dosage sub-group: 91.1%, 92.6%, and 93.7%, respectively） was highest. In 75.0 mg/kg ABZ-CS-MPs group, there were 15 mice with classⅠ（degeneration） and Ⅱ（necrosis） pathological changes of E. granulosus hydatid. The number of mice with class Ⅰ and Ⅱ pathological changes in each dosage ABZ-CS-MPs sub-group and L-ABZ sub-group was more than that of albendazole tablet group（P<0.05）. Plasma concentration of albendazole sulfoxide in 75.0 and 150.0 mg/kg ABZ-CS-MPs sub-groups ［（0.83±0.39）, （0.80±0.5） μg/ml］ were higher than that of L-ABZ sub-groups［（0.34±0.03）, （0.43±0.15） μg/ml］ and albendazole tablet sub-groups ［（0.31±0.02）, （0.40±0.10） μg/ml］（P<0.05）. Compared with 37.5, 75.0, and 150.0 mg/kg albendazole tablet sub-groups ［（0.04±0.02）, （0.07±0.04）, （0.04±0.0） μg/g］, the albendazole sulfoxide concentration in liver tissue was higher in ABZ-CS-MPs sub-groups ［（0.33±0.06）, （0.45±0.31）, （0.50±0.30） μg/g］（P<0.05）. In 37.5 mg/kg dosage sub-group, the albendazole sulfoxide concentration in liver tissue in ABZ-CS-MPs group was higher than that of L-ABZ group ［（0.14±0.19） μg/g］（P<0.05）.  Conclusion  ABZ-CS-MPs can reduce the weight of hydatid cyst and increase the concentration of albendazole sulfoxide in plasma and liver tissue of mice.

Preparation and Antigenicity Analysis of Recombinant Aegyptin-like Protein of Aedes albopictus

LI Xing-Pan, CAO Guo-Mei, XING Cui-Cui, HUA Qian-Qian, ZHANG Liang-Liang, LIANG Shao-Hui

2014, 32(3):  6-193-197. 

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Objective  To clone and express the aegyptin-like protein （alALP） encoding gene from Aedes albopictus salivary gland, and analyze its antigenicity.  Methods  The homology, secondary structure and antigen peptides of alALP and aegyptin protein（GenBank No. ABF18122.1） was analyzed by bioinformatics software tools. Total RNA was extracted from Ae. albopictus salivary gland. The coding region of alALP （GenBank No. AY826121） was amplified by PCR. RT-PCR product was digested with restriction enzyme and ligated into a pGEX-6P-1 vector. The recombinant pGEX-6P-1-alALP plasmid was transformed into E. coli BL21 and induced by IPTG. The recombinant soluble GST-alALP fusion protein was purified with Glutathione Sepharose 4B. The expression product was analyzed by SDS-PAGE and Western blotting. Mice were immunized each with 60 μg purified GST-alALP at every 2 weeks for 3 times, and mouse anti-GST-alALP serum was prepared. Western blotting assay with mice anti-GST-alALP serum and serum of mice exposed to Ae. albopictus bites was used to analyze its antigenicity.  Results  Bioinformatics prediction results showed that alALP and aegyptin had 65.58% homology with a similar secondary structure, and a conservative polypeptide. The product of RT-PCR was 762 bp. The recombinant plasmid pGEX-6P-1-alALP was confirmed by double restriction enzyme digestion, PCR and sequencing. SDS-PAGE and Western blotting analysis showed that the bacteria containing recombinant plasmid pGEX-6p-1-alALP expressed a soluble recombinant fusion protein（Mr 56 000） after being induced with IPTG. Western blotting analysis revealed that GST-alALP protein was recognized by mouse anti-GST-alALP serum and serum of mice exposed to Ae. albopictus bites.  Conclusion  Mature peptide gene of alALP can be expressed in prokaryotic expression system, and the recombinant protein shows antigenicity.

Contrast-Enhanced Ultrasonography of Hepatic Alveolar Echinococcosis in Rats: the Correaltion of Imaging Fratures and Histologic Microvascular Density

SONG Tao, LI Hai-Tao, YANG Ling-Fei, YAO Lan-Hui, WEN Hao

2014, 32(3):  7-200-204. 

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Objective  To correlate the characteristic images of hepatic alveolar echinococcosis （HAE） in rats using contrast-enhanced ultrasonography （CEUS） and microvascular density （MVD） in the surrounding invasion range of HAE lesions.  Methods  Thirty Wistar rats were infected with Echinococcus multolocularis suspension （approximately 800 protoscoleces in 0.2 ml per rat） through abdominal opening injection in liver. Three months after the inoculation, rats with hepatic E. multilocularis infection were examined by conventional and contrast-enhanced ultrasonography. The location, size, shape, boundary, inner echogenicity, and blood flow of the lesions, signal intensity and dynamic enhancement pattern were recorded. The positive rats were sacrificed and their livers were obtained. The structure of HAE lesions was observed by HE staining. Immunohistochemistry staining was performed on the infiltrative region of HAE lesion and the expression of CD34 in the surrounding hepatic parenchyma. Scoring was based on the percentage of positively stained cells and stain intensity. The correlation of MVD and the characteristic images of HAE using CEUS was analyzed.  Results  Twenty-three Wistar rats with hepatic E. multilocularis infection were killed, and 27 HAE lesions was found. The largest diameter of HAE lesion was 2.24 cm, and the average size was（0.97±0.48） cm. The shape of HAE lesions was round, oval, or irregular. HAE lesions presented a complex internal echo pattern. Spot-like color flow signal was observed in the tissues around the lesions, no blood flow signal was observed in HAE lesions. In 25 lesions, a circular rim-like enhancement belt was visualized at the periphery during early arterial phase, and honeycomb enhancement appeared in the other two lesions. The positive expression rate of CD34 in infiltrative zones surrounding HAE lesions was 99.2% （118/119）, with 17.6% （21/119） of strong positive, 73.1% （87/119） of moderate positive, 8.4% （10/119） of weak positive, and 0.8% （1/119） of negative, respectively. In normal liver tissues, the positive expression rate of CD34 was 25.2% （30/119）, no strong positive was found, with 4.2% （5/119） of moderate positive, 21.0% （25/119） of weak positive, and 74.8% （89/119） of negative, respectively. The sonographic infiltrative region in HAE lesion correlated with microvascular density （r=0.238， P＜0.05）.  Conclusion  There is a positive correlation between the circular rim-like enhancement belt surrounding HAE lesions and the microvascular density in the rat model.

Substitution Saturation Analysis of Mitochondrial Cytochrome C Oxidase Subunit 1（COX1）Gene of Angiostrongylus cantonensis

WANG Min, LV Shan

2014, 32(3):  8-205-209. 

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Objective  To determine the substitution saturation of mitochondrial cytochrome c oxidase subunit I（COX1） gene of Angiostrongylus cantonensis.  Methods  One hundred and thirty A. cantonensis adult worms were collected from 33 sampling sites and used to amplify the complete sequence of COX1 gene. The nucleotide diversity，the number of haplotypes and the number of mutations were calculated by DnaSP software after sequence alignment. The distribution of base substitutions was characterized. The level of nucleotide substitution saturation was evaluated by plotting transitions and transversions against pairwise genetic distance.  Results  A total of 130 complete COX1 sequences of 1 577 bp were obtained. One hundred seventy-one nucleotides were found to be variable，resulting in 39 haplotypes with a diversity of 0.8114. The nucleotide diversity was 0.02841. Mutations were evenly distributed along the COX1 gene and did not show significant clustering. The analysis of COX1 gene showed that the amount of substitutions was increasing with the extension of genetic distance，and transitions outnumbered transversions；the increase rate was 0.76，and 0.16，respectively. The substitution rate was markedly different among the three codon positions：the maximum rate was found at the third codon position，followed by the first position，and the second position. The analysis of COX1 gene among 7 members of Metastrongyloidea showed that transversions outnumbered transitions when the genetic distance was more than 0.15. When the number of transitions exceeded 6%，a plateau was reached；while the transvertions increased linearly.  Conclusion  No substitution saturation is found in mitochondrial COX1 gene of A. cantonensis. Therefore， the substitutions at each codon position could be considered in phylogeny analysis.

Anisakis simplex Larvae: Infection Status in Marine Fishes for Sale in Shantou

CHEN Jun-Hua, XU Zhi-Xia, XU Guang-Xin, HUANG Jian-Yun, CHEN Hong-Hui, SHI Shi-Zun, TUN Xiu-Yang, LIANG Jing-Jing

2014, 32(3):  9-212-216. 

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Objective  To investigate the infection status of Anisakis simplex larvae in marine fishes for sale in Shantou.  Methods  Marine fishes were randomly collected from markets in Shantou City from February to December 2013, and then classified. The viscera and muscle of each fish were carefully dissected and thoroughly examined for anisakids. The larvae were examined under a light microscope. The infection rate and intensity of Anisakis simplex larvae were calculated.  Results  A total of 382 fish specimens belonging to 52 species were examined. 42 out of 52 species（80.8%） were found infected by A. simplex larvae. The overall infection rate reached 47.4% （181/382）, and average 5.5 larvae parasitized per infected fish （995/181）. The survival rate of larvae was 100%. The highest infection rate observed was 100% in Scomber australasicus （4/4）， Trachurus japonicus （9/9）, Decapterus maruadsi （8/8）, Lutjanus lutjanus （9/9）, Argyrosomus argentatus （4/4）, Nibea albiflora （4/4）, Nemipterus bathybius （12/12）, Trachinocephalus myops （7/7） and Mene maculata （9/9）, followed by 16/18 in Pneumatophorus japonicus, 6/7 in Lutjanus ophuysenii and 5/6 in Lutjanus fulvus. A. simplex larvae were not detected in 10 fish species, namely, Megalaspis cordyla, Lutjanus argentimaculatus, Lutjanus fulviflamma, Acanthopagrus australis, Acanthopagrus latus, Plectorhinchus nigrus, Dentex tumifrons, Psenopsis anomala, Scatophagus argus, and Seriola lalandi. The infection intensity was the highest in Lutjanus fulvus （21.0 per fish）, followed by Trachinocephalus myops（16.7 per fish）, Saurida filamentosa（14.0 per fish） and Mene maculate（10.1 per fish）. The lowest infection intensity was found in Rastrelliger kanagurta, Kaiwarinus equula, Atule mate, Lutjanus russellii, Plectorhinchus cinctus, Priacanthus tayenus, Branchiostegus argentatus, Branchiostegus albus, Sphyraena pinguis, Formio niger, Trachinotus blochii, Siganus fuscescens and Choerodon azurio（less than 2 per fish）. The highest infection rate（34.3%, 131/382） was found in the mesentery. The infection intensity was highest in pyloric appendage （3.5 per fish）. A. simplex larvae were not found in muscle. The highest infection rate （60.2%, 74/123） was found in fishes with body weight of 100-200 g. The infection intensity was highest in fish with body weight of 301-400 g （7.8 per fish）.  Conclusion  The infection rate of A. simplex larvae is high in marine fishes from Shantou markets.

Identification of Clonorchis sinensis Metacercariae Based on PCR Targeting Ribosomal DNA ITS Regions and COX1 Gene

YANG Qing-Li, SHEN Ji-Qing, JIANG Zhi-Hua, YANG Yi-Chao, LI Hong-Mei, CHEN Ying-Dan, ZHOU Xiao-Nong

2014, 32(3):  10-217-220. 

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Objective  To identify Clonorchis sinensis metacercariae using PCR targeting ribosomal DNA ITS region and COX1 gene.  Methods  Pseudorasbora parva were collected from Hengxian County of Guangxi at the end of May 2013. Single metacercaria of C. sinensis and other trematodes were separated from muscle tissue of P. parva by digestion method. Primers targeting ribosomal DNA ITS region and COX1 gene of C. sinensis were designed for PCR and the universal primers were used as control. The sensitivity and specificity of the PCR detection were analyzed.  Results  C. sinensis metacercariae at different stages were identified by PCR. DNA from single C. sinensis metacercaria was detected by PCR targeting ribosomal DNA ITS region and COX1 gene. The specific amplicans have sizes of 437/549, 156/249 and 195/166 bp, respectively. The ratio of the two positive numbers in PCR with universal primers and specific primers targeting C. sinensis ribosomal DNA ITS1 and ITS2 regions was 0.905 and 0.952, respectively. The target gene fragments were amplified by PCR using COX1 gene-specific primers. The PCR with specific primers did not show any non-specific amplification. However, the PCR with universal primers targeting ribosomal DNA ITS regions performed serious non-specific amplification.  Conclusion  C. sinensis metacercariae at different stages are identified by morphological observation and PCR method. Species-specific primers targeting ribosomal DNA ITS region show higher sensitivity and specificity than the universal primers. PCR targeting COX1 gene shows similar sensitivity and specificity to PCR with specific primers targeting ribosomal DNA ITS regions.

Protein Interaction Site of Toxoplasma gondii Microneme Protein 6 and Aldolase Determined by Site-directed Mutagenesis

ZHENG Bin, YIN Zhi-Kui, ZHAN Xi-Mei

2014, 32(3):  11-221-224. 

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Objective  To identify the protein interaction site of Toxoplasma gondii microneme protein 6 （MIC6） and aldolase by using site-directed mutagenesis.  Methods  Based on Toxoplasma gondii MIC6 gene sequence （GenBank Accession No. AF110270）, the specific primers were designed. Tryptophan（W）-348 of MIC6 C terminus （MIC6C） was mutated to valine（V） via site-directed mutagenesis. MIC6C W/V gene was obtained from cDNA library by PCR amplification and subcloned into pGEX-4T-1. The mutant protein GST-MIC6C W/V was expressed in E. coli, induced by 0.8 mmol/L IPTG, and purified by affinity chromatography. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with T. gondii tachyzoites lysate, and bound proteins were eluted using sample buffer. Bound products were resolved by SDS-PAGE and Western blotting. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with aldolase-His6. After incubation, the resin was washed and subjected to SDS-PAGE.  Results  The MIC6C W/V gene was obtained, and the recombinant plasmid MIC6C W/V/pGEX-4T-1 was successfully constructed. The mutant protein GST-MIC6C W/V was expressed and purified in vitro. SDS-PAGE analysis indicated that GST-MIC6C was co-precipitated with aldolase from T. gondii tachyzoites lysate or aldolase-His6, whereas GST-MIC6C W/V failed to precipitate aldolase from T. gondii tachyzoites lysate or aldolase-His6. Western blotting analysis using anti-aldolase antibody indicated that GST-MIC6C could pull-down aldolase from T. gondii tachyzoites lysate.  Conclusion  Tryptophan （W348） was the interaction site of MIC6 and aldolase in T. gondii.

Intestinal Pathological Changes of Kunming Mice Infected by Cryptosporidium and the Therapeutic Efficacy of Spiramycin on Infected Mice

WANG Dong, ZHANG Yuan-Yuan

2014, 32(3):  12-225-228. 

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Objective  To observe the symptom, disease course of Cryptosporidium-infected mice, and the therapeutic effect of spiramycin on infected mice.  Methods  Seventy Kunming mice were randomly divided into normal control group（A, n=10） and 3 experimental groups（B, C, and D）. Mice in groups B, C, and D（n=20） were immunosuppressed with 5, 7.5, and 10 mg/L dexamethasone in drinking water for two weeks, respectively. The mice were observed and the number of oocysts in fecal sample was counted daily after immunosuppression. Two weeks post immunosuppression, 50% of mice in each group were sacrificed, and small intestine was removed for observation of parasitic site. The pathological changes of mucous membrane were observed under microscope, and sIgA level in intestinal fluid was determined. Immunosuppression was withdrawn in the rest mice, and after two weeks unrecovered mice were divided into treatment group（n=6） and control group（n=7）. Mice in treatment group were each given 2 mg/d spiramycin for 10 d. Each mouse in control group was given same amount of normal saline. The mice were observed and the oocysts shedding in fecal pellets were counted after treatment.  Results  On the 6th day post immunosuppression, Cryptosporidium sp. positive fecal samples were found in the experimental groups. The number of oocysts per gram of feces in groups D（70.3±4.0） and B（31.9±2.4）reached a peak on the 12th day and 10th day post immunosuppression, respectively（P＜0.05）. The conspicuous enteron symptom was observed in each experimental group. Cryptosporidium parasitized mainly in upper jejunum. Pathological examination of intestinal mucous membrane showed that swollen mucous membrane and hemorrhages were observed in group D, and less inflammatory cell occurred in groups B and C. sIgA level in intestinal fluid of experiment mice descended, there was a statistical significance between groups D  [（2.7±0.6）μg/ml], C  [（3.2±0.8） μg/ml], B [（4.9±1.3）μg/ml] and A [（6.1±1.2）μg/ml] （P＜0.05）. After withdrawal of immunosuppression, out of 30 positive mice, symptoms including diarrhea and loose stools improved in 17 mice. After treated with spiramycin, status of the mice got improved, and there was statistical significance in the level of oocyst shedding between treatment group（0）and control group（11.3±8.1）（P＜0.05）.  Conclusion  The level of oocyst shedding, disease course, and  pathological change of intestinal mucosa membrane are closely related to immune status of Cryptosporidium-infected mice. Spiramycin is effective in treating of Cryptosporidium infection.

Endemic Situation and Control Progress of Taeniasis in Western China

LONG Chang-Ping, JIAN Ying-Jun, LI Tiao-Yang, FU Qing, WANG Qiang, XIAO Ning

2014, 32(3):  13-229-233. 

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Taeniasis, caused by Taenia species, is one of the common zoonoses in China, particularly in the western region of China. Up to now, not enough attention has been given in the high prevalence and high burden of the diseases. In order to study the endemic patterns and control strategies of taeniasis, a series of epidemiological investigations, molecular researches and pilot control activities have been conducted in recent years. This paper reviews the relevant publications in taeniasis research over the last 10 years.

Progress on IL-27 in Immunity to Important Protozoan Parasitic Infections

TONG Xin-Xin, LV Fang-Li

2014, 32(3):  14-234-238. 

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Parasitic infection can stimulate a series of immune responses and lead to changes in cytokines. This paper focuses on the progress on the role of IL-27 in immunity to some protozoan infection, caused by Toxoplasma gondii, Leishmania, Plasmodium, and Trypanosoma cruzi.

Identification of Three Common Sandfies in Southern Xinjiang with Multiplex PCR

ZHOU Zheng-Bin, ZHANG Yi, ZHU Huai-Min, SHI Wen-Qi, JIN Chang-Fa

2014, 32(3):  15-185-187. 

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Based on the variable part of mtDNA COⅠ gene sequence， a multiplex PCR method was developed for the identification of the three common sandflies（Phlebotomus longiductus， Ph. wui， and Ph. alexandri） in southern Xinjiang. The results demonstrated that this multiplex PCR method was reliable， and could be used to identify the three Phlebotomus species. The PCR product of COⅠ gene from Ph. longiductus， Ph. wui and Ph. alexandri was 248, 632， and 395 bp， respectively.

Seroepidemiological Survey on Echinococcosis in Primary School Pupils of Ganzi Tibetan Autonomous Prefecture of Sichuan Province in 2012

FAN Dao-Yong, LIU Tao, WANG Zai-Yue, XIE Xiao-Lin, WANG Li

2014, 32(3):  16-197-199. 

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In May 2012, 11 echinococcosis-endemic counties in Ganzi Tibetan Autonomous Prefecture of Sichuan were chosen, and two primary schools were randomly selected in each county from urban and non-urban area. Serum anti-echinococcus IgG was detected by ELISA. Among 5 171 sampled children, the sero-positive rate was 0.8%（43/5 171）. The rate in males and females was 0.7%（17/2 538） and 1.0%（26/2 633）, respectively（χ2=1.581，P＞0.05）. The sero-positive rate in urban schools and non-urban schools was 0.7%（14/2 078） and 0.9%（29/3 093）, respectively（χ2=1.050, P＞0.05）. The positive rate in the minorities（1.0%, 41/4 273） was higher than that of the Han nationality （0.2%, 2/898）（χ2=4.884, P<0.05）. Compared with 2010, 2011, the total positive rate of children in 2012 declined significantly（χ2=112.945, P<0.01）.

PCR-based Genotype Classification of Blastocystis hominis Isolates from College Students of Guangxi

ZHAN Ting-Zheng, LIU Teng, DAN Huan-Huan, HE Shan-Shan, YAN Hui, LIU Deng-Yu

2014, 32(3):  17-209-211. 

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Fifty-three Blastocystis hominis isolates were separated from the fecal specimens of carriers in college students from Guangxi and cultivated in vitro， and the genetic DNA was extracted. All the isolates were genotyped by PCR using seven pairs of known sequence-tagged site（STS） primers. The results showed there were five subtypes in the 53 isolates. Subtype 3 was the most popular one （32.1%, 17/53）, followed by subtype 7 （9.4%, 5/53）. Subtypes 1 （7.6%, 4/53）， 4 （7.6%, 4/53）， and 6 （1.9%, 1/53） were detected， while subtypes 2 and 5 were not detected. The genotypes of the other 22 isolates were unknown which were negative to all the STS primers.

Construction and Identification of the Bifidobacterium Expression System pGEX-TSOL18/B. longum of Taenia solium

ZHOU Bi-Ying, LIU Mei-Chen, HE Li-Fang

2014, 32(3):  18-239-241. 

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The TSOL18 gene of Taenia solium was synthesized and cloned into Escherichia coli-Bifidobacteria shuttle vector pGEX-1λT. The recombinant plasmid pGEX-TSOL18 was transformed into Bifidobacterium longum with electroporation. The recombinant plasmid containing TSOL18 gene was identified by restriction endonuclease analysis， PCR and DNA sequencing. The length of synthesized TSOL18 gene was 393 bp. The results indicated that the Bifidobacteria expression system pGEX-TSOL18/B. longum was successfully constructed.

In Vitro Effect of Osthole on Ultrastructure of Giardia lamblia

LI Wen-Chao, GU You-Fang, LIU Chang, WU Na, LUO Wen-Wu, GONG Feng-Tao, LI He, LI Jian-Hua, ZHANG Xi-Chen

2014, 32(3):  19-242-244. 

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Giardia lamblia trophozoites were cultivated axenically in TYI-S-33 modified medium containing 1.345 mg/ml of osthole(24 h IC50）. The parasites were observed by scanning and transmission electron microscopes after treated with osthole for 24 h. The surface of the trophozoites treated with osthole was rough. The surface of ventral sucker and median body had obvious lesions, the cell membrane was damaged and the content spilled out. There were a lot of vacuoles in the cytoplasm. And the nuclear was severely deformed with a serrated edge and marginated nuclear chromatin. The microtubules of sucker had partially disintegrated.

Echinococcus granulosus Infection in Dogs and Livestock from Xinjiang Production and Construction Corps

HAN Fei, WANG Bing-Quan, WANG Li-Jie, XIONG Jun, XI Yi, WU Li-Wen, MA Fu-Rong, LI Fan-Ka

2014, 32(3):  20-245-246. 

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The prevalence of Echinococcus granulosus infection in dogs and livestock was investigated in Xinjiang Production and Construction Corps by stratified random sampling. A total of 5 391 dog feces were detected by double antibody sandwich ELISA, and the positive rate of dog coproantigen was 0.69%（37/5 391）. The livestock were subjected to necropsy, inspection and palpation. The prevalence of E. granulosus infection in livestock was 3.88%（431/11 122）.