Table of Content

30 April 2012, Volume 30 Issue 2

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Ultrastructural Observation on Nymphal Armillifer sp. by  Scanning Electron Microscopy and Phylogenetic Analysis Based on 18S rRNA

LI Jian1，SHI Yun-liang1，SHI Wei1，FANG Fang1，ZHOU Qing-an1，

2012, 30(2):  1-81-85. 

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  Objective  To observe the ultrastructure of nymphal Armillifer sp. isolated from Macaca fascicularis by using scanning electron microscope (SEM), and analyze the phylogenetic relationships based on 18S rRNA gene sequences.  Methods  The parasite samples stored in 70% alcohol were fixed by glutaraldehyde and osmium peroxide. Ultrastructural characters of those samples were observed under SEM. Amplification and sequencing of the 18S rRNA gene were performed following the extraction of total genome DNA. Sequence analysis was performed based on multiple alignment using ClustalX1.83, while phylogenetic analysis was made by Neighbor-Joining method using MEGA4.0.  Results  The nymphs were in cylindrical shape, the body slightly claviform tapering to posterior end. Abdominal annuli were gradually widened from anterior to posterior parts, the 12th-13th abdominal annuli of which were similar in width. The annuli ranged closer in the front half body, whereas in the latter part there were certain gaps between them. The circular-shaped mouth located in the middle of head ventrally. Folds were seen in inner margin of the mouth with a pair of curved hooks on both sides above it which practically disposed in a straight line. Two pairs of large sensory papillae were observed symmetrically over the last thoracic annulus of cephalothoraxs lying below the outer hook, and the first abdominal annulus was near the median ventral line. The number of abdominal annuli was 29, not including 2 incomplete terminal annuli. Rounded sensory papillae were fully distributed on the body surface, except the dorsal side of head and the ventral part of the terminal annulus. Agglomerate-like anus opening was observed at the end of ventral abdominal annuli and distinctly sub-terminal. These morphological features demonstrated that the nymphs were highly similar with that of Armillifer moniliformis Diesing, 1835. A fragment of 18S rRNA gene (1 836 bp)sequences was obtained by PCR combined with sequencing, and was registered to the Gen-eBank database with an accession number HM048870. The phylogenetic tree indicated that A. moniliformis, A. agkistrodontis and A. armillatus were at the same clade with a bootstrap value at 95%, and A. moniliformis and A. agkistrodontis were solo at a clade with a bootstrap value of 75%.  Conclusion  The nymphs isolated from Macaca fascicularis are identified as A. moniliformis temporarily.

Cloning，Expression and Antigenicity Analysis of Phosphoglycerate Mutase 2 Gene of Toxoplasma gondii

YAN Li-Tian1, WANG Fen2, MENG Xiao-Li2, WANG Hai-Long2, LIU Gong-Li2, SHEN Jin-Yan2, YAN Guo-Rong2 *

2012, 30(2):  2-86-89. 

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Objective  To clone and express the phosphoglycerate mutase 2 (PGAM2) gene of Toxoplasma gondii, and analyze the antigenicity of the recombinant protein.  Methods  Total RNA was extracted from T. gondii tachyzoites of RH strain and reversely transcribed into cDNA. TgPGAM2 gene was amplified by PCR and cloned into pET30a(+) vector. The constructed pET30a(+)-TgPGAM2 was transformed into E. coli DH5α first and selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The correct plasmid was transformed into E. coli BL21 for expression induced by IPTG and the recombinant protein was further analyzed through SDS-PAGE followed by Coomassie brilliant blue staining. Western blotting assay with rabbit anti-T. gondii serum was used to analyze its anti-genicity.  Results  The length of PCR product was about 750 bp and the recombinant plasmid pET30a(+)-TgPGAM2 was successfully constructed. The results of SDS-PAGE and Western blotting revealed that the relative molecular weight (M_r) of the soluble recombinant protein was approximately 30 000 and could be recognized by rabbit anti-T. gondii serum.  Conclusion  The soluble TgPGAM2 protein has been expressed in the prokaryotic expression system and maintains its antigenicity.

Establishment and Evaluation of Colloid Gold Labeled Immuno-hromatographic Strip Test for Rapid Diagnosis of  Alveolar Echinococcosis

GAO Chun-Hua, DAN Feng, HONG Dun-Yun*, YANG Yue-Chao, SHU Hui-Hui

2012, 30(2):  3-90-94. 

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Objective   To establish and evaluate a colloid gold immunochromatographic strip test for the diagnosis of alveolar echinococcosis.  Methods  Total RNA was prepared from Echinococcus multilocularis protoscoleces collected from Xinjiang Uygur Autonomous Region. Em18 gene was obtained by reverse transcription-polymerase chain reaction(RT-PCR). The PCR product was sequenced and cloned into pGEX-3X vector. The recombinant plasmid was expressed and induced by isopropyl-β-D-thiogalactopyranoside (IPTG) to obtain recombinant protein. The anti-human IgG monoclonal anti-bodies was conjugated with colloid gold as detecting reagent; the recombinant Em18 antigen and goat anti-mouse IgG were immobilized on nitrocellulose in proper position. The prepared immunochromatographic strip was evaluated using serum samples from patients with alveolar echinococcosis（56），cystic echinococcosis（87），cysticercosis（30），schistosomiasis japonica（10），toxoplasmosis（10）and healthy subjects（50）. Comparison between the immunochromatographic strip test and ELISA was made by kappa statistics.  Results  Sensitivity detected by the immunochromatographic strip test was 92.9%（52/56）. The cross-reactivity to cystic echinococcosis and cysticercosis was 9.2%（8/87）and 3.3%（1/30）, respectively. There was no cross reactivity with schistosomiasis japonica and toxoplasmosis. 4 samples out of 50 healthy people showed false positive reaction. The overall specificity was 93.0%（174/187）. Sensitivity and specificity both showed no statistical difference between immunochromatographic strip test and ELISA. High degree of agreement was observed between the strip test and ELISA (κ=0.98).  Conclusion   The developed immunochromatographic strip test using recombinant Em18 antigen as coated antigen is a sensitive，specific，simple and rapid assay for diagnosing alveolar echinococcosis.

Prevalent Trend of the Infection of Soil-transmitted Nematodes in Fujian Province

LI Chi-Sha*, CHEN Bao-Jian, ZHANG Rong-Yan, CHENG Yao-Zhou, LIN Chen-Xin, LIN Kai-Qian, LI Yan-Rong, FANG Pan-Tan, ZHENG Guo-Bin, JIANG Dian-Wei

2012, 30(2):  4-95-99. 

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Objective  To understand the endemic situation of soil-transmitted nematodes in Fujian Province.  Methods  According to the national guidelines, the survey spots were determined by clustered random sampling in nine prefectures (cities) of Fujian Province from 2007 to 2009. Residents of 3 years old and above were investigated. The eggs of roundworm, hookworm and whipworm in feces were checked by Kato-Katz method. Eggs per gram (EPG) in feces were calculated. The gender, age and education status of the investigated subjects were recorded. The results were evaluated in comparison to those in 1992 and 2003.  Results  Altogether 93 833 residents in 610 villages of 184 towns from 49 counties were investigated. The overall infection rate of soil-transmitted nematodes was 10.14%(9 511/93 833), decreased by 86.88% and 71.84% compared to that in 1992 and 2003, respectively. The prevalence of roundworm, hookworm and whipworm was 1.32%(1 234/93 833), 7.31%(6 863/93 833) and 1.73%(1 622/93 833), respectively. The average EPG of roundworm, hookworm and whipworm was 9 556, 526 and 156, respectively. The prevalence in males and females was 9.48%(4 385/46 246) and 10.77%(5 126/47 587), respectively, with a statistical difference ( χ²＝42.84, P＜0.01). There was also a statistical difference among the age groups ( χ²＝1 626, P＜0.01). The higher education level of the people, the lower prevalence ( χ²＝1 107， P＜0.01).  Conclusion  The prevalence of soil-transmitted nematodes is on a downward trend in Fujian Province, but remained high in the underdeveloped areas. The hookworm infection rate is higher than the average of the nation.

Study on the Correlation between Land Use and Cover Change and Malaria Transmission in the Areas along the Yellow River and Huai River

ZHANG Shao-Sen, ZHOU Shui-Sen*, SHANG Lin-Hua, HUANG Fang, ZHENG Xiang

2012, 30(2):  5-102-108. 

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Objectives   To study the major ecologic factors influencing the re-emergence of malaria in the areas along the Yellow River and Huai River by analyzing the relationship between the malaria incidence and the land use and cover change (LUCC).  Methods  The data of annual parasite incidence (API) and LUCC in the counties of  Huaiyuan, Yongcheng and Tongbai in 1990-2006 were collected retrospectively. Considering the hysteresis effect of LUCC played on malaria transmission, analysis of LUCC in these counties were conducted based on the data of 1990-1995 and 1996-2000, while the API data in 1996-2000 and 2001-2005 were used to analyze the trends and changes of malaria incidence. The correlation and trend analyses were conducted between LUCC and malaria incidence change in the periods.  Results  The LUCC in 1990-1995 in Tongbai County was significant, being 3 265.79% in the farmland and -90.42% in paddy field. The increase of malaria incidence in Tongbai was also significant in 1996-2000, the change rate was 2 799.70%, showing a positive correlation. The LUCC of other 2 counties in 1990-1995 was -0.27% and -0.78%, respectively, while the rate of malaria incidence change in 1996-2000 was 206.25% and 0.00%. The LUCC of the 2 counties in 1996-2000 was -0.08% and -0.50%, while the rate of malaria incidence change in 2001-2005 was 153.22% and 2 500.00%, respectively. It indicated that there was no significant relationship between malaria re-emergence and LUCC in Huaiyuan and Yongcheng Counties.  Conclusion  It seems that the LUCC shows no significant impact on malaria re-emergence in areas along the Yellow River and Huai River since 2001, though there was a positive correlation between the two in Tongbai County in the late 1990s.

Immunoscreening of Schistosoma japonicum Egg cDNA Library and Identification of Positive Clones

LEI Yan1, XU Bin2, JU Chuan2, MO Xiao-Jin2, CHEN Shen-Bei2, FENG Zheng2, WANG Xiao-Ning1, HU Wei-1

2012, 30(2):  6-109-115. 

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Objective   To obtain promising antigens for diagnosis or therapeutic efficacy assessment of schistosomiasis japonica.  Methods  At an endemic area in Anqing of Anhui Provinces, Kato-Katz method (six slides from two consecutive stool samples) was used to examine schistosome eggs and serum samples of 10 egg-positive patients were collected. The EPG of the ten patients, aged 13 to 64, was among 4 to 172. The patients were then treated with praziquantel 60 mg/kg for two days. Sera of the same patients were collected again at six months post treatment. The egg cDNA library was immunoscreened with the pre- and post-treatment serum samples， and the positive clones were classified according to different reactions. The inserted fragments of positive clones were sequenced and analyzed for their homology through BLAST program. The amino acid sequences of the proteins were deduced from the gene sequences, and their structures and functions were predicted by bioinformatics tools.  Results  For the first round， 75 positive clones were screened from the egg cDNA library. Among them, 21 clones showed stronger reaction with the pre-treatment sera， and 8 clones showed stronger reaction with the post-treatment sera. According to the intensity of the reaction， types and the length of inserted fragments， 14 positive clones were selected for further study. Most of the clones belonged to miracidial antigen family， one clone was significantly homologous to the calcium-binding EF-hand， a domain-containing protein (score=143)， one was highly homologous to the cell wall integrity and stress response component 1 precursor (score=487)， and another four were homologous to the hypothetical protein. Conclusion  Twenty nine positive clones screened from the egg cDNA library show different reactions with the sera of schistosome-infected patients, before and after praziquantel treatment.

Pathogen Identification and Clinical Diagnosis for One Case Infected with Babesia

TAO Li-Nong1, RUAN Wei1, CENG Chang-You2, LI Jie-Huo2, ZHANG Han1, LEI Yong-Liang2, LIU Qiao-Shi1, CHEN Hua-Liang1

2012, 30(2):  7-118-121. 

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Objective   To identify the pathogen and make diagnosis on a case who was misdiagnosed as malaria.  Methods  Clinical and epidemiological information of the suspected case was collected. Blood samples during hospitalization were collected and examined microscopically. Genomic DNA from the blood samples was amplified by Babesia 18S RNA genus- and species- specific primers, respectively, and the amplified products were used in sequencing and BLAST sequence analysis.  Results  The case had a fever over 20 days repeatedly with anaemia (RBC 2.59×1012，HB 5.5 g/L) and hepatosplenomegaly. The unidentified parasites were found in the bone marrow and blood smear after Giemsa staining. Epidemiological information revealed that this case had a history of blood transfusion and tick bites. 1 625 bp and 449 bp band generated by PCR amplification from blood sample using Babesia genus- and species-specific primers, and the sequence homology was 99% in comparison to Babesia microti (AB241632) with BLAST analysis. Conclusion  The clinical information, epidemiological history, and the PCR identification confirm the diagnosis of Babesia microti infection.

Investigation on Blood-sucking Habit of Anopheles (Diptera∶Culicidae) Using Multiplex Polymerase Chain Reaction in Malaria-Endemic Area of Chayu County, Tibet

GUO Chao-Hua1, ZHOU Shui-Sen1 *, HUANG Fang1, ZHENG Xiang1, WU Song2, ZHOU Hua-Yun3, ZHUO Ma-Yang-Jin4

2012, 30(2):  8-122-126. 

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Objective   To determine the blood-sucking habit of anopheline by mosquitoes bloodmeal identification in malaria endemic area Chayu County, Tibet Autonomous Region.  Methods  Three villages with different bio-enviroments were selected as the investigation spots. Light traps were set up outdoor and in livestock sheds from sunset （20 ∶ 00） to sunrise （8 ∶ 00） in 3-4 consecutive nights to collect mosquitoes. The trapped anophelines were counted and identified according to morphological criteria and multiple PCR method. A PCR-based methodology according to the mtDNA-cytb variations was used in different mammal hosts to identify bloodmeal sources in engorged mosquitoes. The human blood index (HBI) was assessed to determine the range of hosts.  Results  Among 1 442 anopheline mosquitoes collected by 108 lighttraps on 13 nights. 1 436（99.6%） belonged to Anopheles maculatus complex, with 85.5% An. pseudowillmori and 14.5% An. willmori. Positive bloodmeal identification was found from 168（83%） of 202 field-collected engorged mosquitoes. The crude HBI of An. pseudowillmori and An. willmori were 0.35 and 0.29, respectively.  Conclusion  An. pseudowillmori and An. willmori are both zoophilic and anthropophilic, and An. pseudowillmori shows a higher HBI.

Geographical Distribution of Echinococcosis among Children in Qinghai Province

CA Hui-Xia1, GUAN Ya-Yi1*, WANG Hu2, WU Wei-Beng1, HAN Xiu-Min2, MA Xiao2, WANG Li-Yang1, LI Dun1

2012, 30(2):  9-127-130. 

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Objective  To analyze the epidemiological status of echinococcosis among children in three different zones of Qinghai Province.  Methods   B-ultrasound and ELISA were used in the survey to evaluate echinococcus infection among 6～15 year old children in the three zones，namely，Qinghai southern plateau，Qilian mountain-Hehuang valley and Chaidamu basin.   Results  The prevalence of echinococcus infection scanned by B-ultrasound and sera positive rate detected by ELISA in children were 1.5%(320/20 730)and 6.5%(1024/15 762）respectively，and the prevalence of cystic echinococcosis and alveolar echinococcosis by ultrasound were 1.0%（206/20 730）and 0.6%（114/20 730），respectively. The prevalence and sera positive rate were 9.5%（707/7 453)and 2.3%（269/11 618） in Qinghai southern plateau, 3.8%（289/7 544) and 0.6%（50/8 275) in Qilian mountain-Hehuang valley, and 3.7%（28/765)and 0.1%（1/837) in Chaidamu basin, respectively. The southern plateau showed the highest prevalence, with a significant statistical difference among the three areas(P<0.01). The prevalence of alveolar echinococcosis was 1.0%(114/11 618)in the southern plateau, but no alveolar echinococcosis patients were found in other two areas. Correlation analysis showed that the prevalence increased with the altitude (r _s-e=0.96，P<0.05，R ²=0.93；r _p-e=0.82，P<0.05，R ²=0.67).  Conclusion  The prevalence of echinococcosis among children shows an apparent geographical difference in Qinghai Province.

Study Progress on Mefloquine against Schistosomes and other Helminths

XIAO Shu-Hua, XUE Jian

2012, 30(2):  10-131-138,145. 

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【Abstract】  In recent years，antimalarial drug mefloquine，an amino alcohol compound，has been found to exhibit potential effect against schistosomes. The feature of antischistosomal properties of mefloquine is that the drug possesses similar killing effect against various development stages of juvenile and adult schistosomes. This paper summarizes the recent three years′ progress in experimental studies of mefloquine against schistosomes and other helminthes.

Progress on Hydrolases-targeting Antiparasitic Prodrugs

WEN Ai-Dan, ZHANG Hao-Bing*

2012, 30(2):  11-139-145. 

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【Abstract】  Prodrug strategies have been used for drug optimization to overcome the drawbacks in pharmaceutics, pharmacokinetics and pharmacodynamics. Most enzymes involved in prodrug biotransformation are hydrolases，in which esterase and amidase have been widely researched. This review summarizes the recent progress in antiparasitic prodrugs based on both targets.

Research Progress on Cystatin of Parasitic Nematodes

TAO Ju-Xia, FU Bao-Quan*

2012, 30(2):  12-146-151. 

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【Abstract】  Cystatins are reversibly tight-binding natural inhibitors of cysteine proteases. Cystatins in parasitic nematodes not only have the unique inhibition activity on cysteine proteases but also modulate the host immune response and have an important role in the immune evasion from host response and the adaptation to parasitism. This article reviews the classification, structure characteristics and function mechanism of cystatins, and the research progress on cystatins in parasitic nematodes.

Chinese Invention Patent about Anti-parasitic Drug Albendazale

DIAO Jun, GAO Hui-Jing, WANG Jian-Hua*

2012, 30(2):  13-152-156,159. 

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 【Abstract】   This paper summarizes authorized Chinese invention patents about albendazole and shows research status of albendazole from patents. It can provide data for persons involved in the relevant research activities on the drug.

Difference Analysis among Majors in Medical Parasitology Exam Papers by Test Item Bank Proposition

GU Lin-Zhi1, MA Ya-Jun2 *, CAO Yi2, JIAN Feng2, LI Xiang-Yu2

2012, 30(2):  14-160-163. 

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【Abstract】   The quality index among“Medical Parasitology”exam papers and measured data for students in three majors from the university in 2010 were compared and analyzed. The exam papers were formed from the test item bank. The α reliability coefficients of the three exam papers were above 0.70. The knowledge structure and capacity structure of the exam papers were basically balanced. But the α reliability coefficients of the second major was the lowest，mainly due to quality of test items in the exam paper and the failure of revising the index of test item bank in time. This observation demonstrated that revising the test items and their index in the item bank according to the measured data can improve the quality of test item bank proposition and reduce the difference among exam papers.

Investigation on Taenia sp. Infection in Midu County of Yunnan Province

FANG Wen1 *, LIU Hong-Kun1, LI Ke-Rong1, LUO Hua2, XU Xin3, CHEN Feng1, LI Rong1, LIU Ji-Bing2, HUANG Meng-Hao1, LI Su-Mei1

2012, 30(2):  16-116-117,121. 

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【Abstract】  The current status and species of Taenia sp. were investigated in Midu County by sedimentation method to examine eggs of Taenia sp. in stool, questionnairing as well as deworming by areca-pumpkin seeds in October-December, 2010.  The infection rate of Taenia sp. was 15.7%（65/414）. Among the positives, it was fairly high in the age groups of 40- and 50-, being 24%(21/85)and 26%(15/57), respectively. 26 cases with positive stool examination and 47 cases with a history of discharging proglottids were treated. Adult worms were collected from all 26 egg positive cases and 23 persons discharging proglottids. The highest number of adult worms expelled was 11 in a woman, 2 worms from another villager, but only one worm each from all other cases. 15 tapeworms with scolex and mature proglottids were examined and morphologically identified as T. asiatia. The high prevalence was related to the residents′ dietetic habits (eg. eating raw pork and liver), behaviour (eg. defecating in field), and the egg-contaminated environment (eg. by untreated feces).

Impact of Toxoplasma gondii on the Proliferation  and Apoptosis of Tumor Cell Lines

WU Xin1, SUN Li2, ZHANG Li3, LIU Zheng-Quan4, LUO Jiang2, ZHANG Lin-Xi2 *

2012, 30(2):  17-157. 

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【Abstract】 MCF-7，DU-145，EC109 and A549 tumor cells （5×104/ml） at mid-exponential phase were treated for 24 h with different concentrations of Toxoplasma gondii tachyzoites（1×10⁴/ml-32×10⁴/ml）. The measurement of tumor cell growth inhibition rate was performed by using CCK-8 kit. The tachyzoites （1×10⁴/ml-4×10⁴/ml） were co-cultured with A549 tumor cells for 24 h. Flow cytometry techniques was applied to investigate the expression of p53 and Bcl-2. The results showed a remarkable inhibitory effect of the parasites on tumor cell proliferation，which presented a dosage dependence. MCF-7，DU-145，EC-109 and A549 cell lines treated with 1×10⁴/ml T. gondii tachyzoites presented an inhibition rate of 19.7%，4.5%，28.0%，and 20.8%，respectively. There was a significant difference between experimental groups and the control （P<0.05）. T. gondii induced cell apoptosis in A549 tumor cells. p53 and Bcl-2 gene expression was respectively up-regulated and down-regulated.

A Duplicate Staining Method for Permanent Specimen of  Trichinella spiralis Encapsulated Larvae

LI Dan1, YANG Ding1, PI Ben-Wei1, NIU Li-Na1, ZHANG Ying1, WANG Guo-Yang2 *

2012, 30(2):  18-164-封三. 

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【Abstract】 With single staining method，Trichinella spiralis encapsulated larvae specimens were fixed with formaldehyde alcohol acetic acid fixative solution，and stained with alcohol borax-carmine staining solution（4% borax solution 100 ml，carmine 1 g，and 70% alcohol 100 ml）. With duplicate staining，the encapsulated larvae specimens were fixed with formaldehyde alcohol acetic acid fixative solution，and double stained with alcohol borax carmine staining solution and fast green staining solution（fast green 0.1 g，95% alcohol 100 ml）. The results showed that with single staining，it was not clear-cut between the cyst and muscle cells although the larva was differentiable，while with duplicate staining，the larva，cyst and muscle cells were distinguished more clearly.