Table of Content

30 August 2011, Volume 29 Issue 4

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Cloning，Expression and Immunologic Identification of C31B8.8 Gene of Caenorhabditis elegans

SUN Juan, LI Zheng-Yu, HE Han-Jiang, LV Zhi-Ti, TUN Zhong-Dao

2011, 29(4):  1-241-246. 

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Objective   To clone and express C31B8.8 gene of wild-type Caenorhabditis elegans， and study the immunological characteristic of the recombinant protein.   Methods   Total RNA was extracted from cultivated C. elegans and reversely transcribed into cDNA. C31B8.8 gene was amplified by PCR and cloned into pMD-18T vector for sequencing. The accurate sequence was subcloned into the expression vector pET-30a with （His） 6-tag. The recombinant plasmid was transformed into E. coli BL21 and followed by expression of the protein induced by IPTG. The recombinant protein was identified by using matrix-assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF-MS） and Western blotting. 10 BALB/c mice were randomly divided into C31B8.8 immunized group and PBS+adjuvant group. Mice in C31B8.8 immunized group were immunized with 40  g of purified C31B8.8 antigen formulated in Freund′s adjunvant. Mice in PBS+adjuvant group received only adjuvant emulsified with PBS. All the mice received four immunizations every week with the same dose of antigen. Serum samples were collected at preimmunization and certain time after immunization and the antibody titer was analyzed by ELISA. The recombinant C31B8.8 protein and soluble components of Angiostrongylus cantonensis fourth stage larvae were identified by Western blotting.  Results   The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing. MALDI-TOF-MS and Western blotting analysis showed that the recombinant C31B8.8 protein was the target protein. Compared with PBS+adjuvant group， mice immunized with purified protein C31B8.8 produced higher level of IgG. The antiC31B8.8 serum recognized recombinant C31B8.8 protein， and reacted with soluble antigens of A. cantonensis fourth stage larvae.  Conclusion   C. elegans C31B8.8 gene shows certain immunogenicity and immunoreactivity， and the soluble antigens of A. cantonensis fourth-stage larvae can react with anti-C31B8.8 serum.

L-Arginine Enhances Th1 Immune Response against Plasmodium yoelii 17XL Infection in DBA/2 Mice via Activation of Dendritic Cells

PAN Yan-Yan, Liu-Jun, Li-Ying, Yan-Juan, Cao-Ya-Meng

2011, 29(4):  2-247-251. 

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Objective  To investigate the effect of L-arginine （L-Arg） during blood-stage infection by P.y17XL in DBA/2 mice. Methods  DBA/2 mice were divided into 2 groups， 20 mice in each group. Mice were respectively administered with L-Arg (1.5 g/kg, L-Arg group) and normal saline (control group) 7 days before they were infected intraperitoneally by 1×106 pRBC. Parasitemia were detected by Giemsa stained thin-smear microscopy and survival rate were monitored daily. Flow cytometry was introduced to detect the subsets of splenic CD4+CD69+T cells, F4/80+CD36+ macrophages, myeloid dendritic cells(mDCs) (CD11c+CD11b+), plasmacytoid dendritic cells (pDCs) (CD11c+B220+) on day 3, 5 post infection (p.i.). The levels of IFN-γ and NO in the supernatant of splenocytes culture were detected by ELISA and Griess reaction, respectively.  Results   Pretreatment of mice with L-Arg significantly decreased the parasitemia from 45% to 20% and shortened selfcure time from 22 d to 20 d after infection. The level of F4/80+CD36+ macrophages [(29.61±0.47)%], IFN-γ [(485.84±39.31) pg/ml], CD4+CD69+ T cells [(7.3±0.68)%],  NO [(42.51±1.32)  μmol/L], mDCs(CD11c+CD11b+) [(5.51±0.87)%] and pDCs(CD11c+B220+) [(5.60±0.85)%] in L-Arg group was higher than those in control group [(36.46±1.33)%,  (767.86±20.56) pg/ml, （11.27±0.97）%,  (78.66±2.89) μmol/L, (10.02±0.37)%] and (9.01±0.53)%, respectively].  Conclusion  L-Arg enhances Th1 immune responses during the early stage of P.y17XL infection in DBA/2 mice via the activation of DCs.

Conjugation of Plasmodium falciparum Pfs25 to Pseudomonas aeruginosa ExoProtein A with Different Chemical Linkers

QIAN Feng

2011, 29(4):  3-254-257. 

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Objective   To conjugate Plasmodium falciparum Pfs25 to recombinant Pseudomonas aeruginosa ExoProtein A (rEPA) and test the effect of conjugation using different chemical linkers.  Methods   The Pfs25 was thiolated by NAHT (DL-N-acetylhomocysteine thiolactone)， whereas the carrier protein rEPA was modified by Sulfo-EMCS， EMCH，SBAP and Sulfo-SIAB respectively. The Pfs25-rEPA conjugates were formed by incubating the thiolated Pfs25 with modi-fied rEPA under certain conditions. Coomassie blue stained SDS-PAGE was performed to examine the results of the conjugation.  Results   The Pfs25 was successfully conjugated to the carrier protein rEPA by the linkers used in this study. For the addition of a chemical group onto the protein， the reaction between the groups of primary amine and NHS (N-hy-droxysuccinimide) ester was more efficient than the reaction between the groups of carboxyl and hydrazide with EDC as a crosslinker; for the formation of a conjugate， the reaction between the groups of maleimide and free sulfhydryl was more efficient than the reaction between the groups of haloacetyl and free sulfhydryl.   Conclusion   The Pfs25 can be conjugated to the rEPA by the chemical linkers with different conjugation efficiency and coupling proteins.

Acaricidal Activities of the Water Extract of Xiushan Sea Mud against Human Demodex Mites

HU Ye, Yang-Chao, Xin-Jian-Mei, Luo-Gong-Yu

2011, 29(4):  4-258-263. 

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Objective   To observe the acaricidal effect of the water extract of Xiushan sea mud on Demodex brevis and D. folliculorum.   Methods   D. brevis and D. folliculorum were obtained with modified scraping method. Each sample contained 30 or more alive Demodex which were evenly spread on the slides with 200 μl water extract of Xiushan sea mud at a concentration of 2.5，2.0，1.5，1.0 and 0.5 kg/L，respectively. With 10% sulfur emulsion as positive control and saline as blank control，each experiment group was carried out at the same time. Under room temperature of 20 ℃ and relative humidity of 70%，parasite death number was recorded at 5 min, 10 min, 20 min, 40 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. The acaricidal rate was measured and calculated by counting method. Demodex activity and morphology were observed using microscope.   Results   1.0～2.5 kg/L of the water extract of Xiushan sea mud increased the activities of both kinds of Demodex. D. brevis showed shrinkage, deformation, twisting and the bodies became shorter. The digesting vasoconstrictions of D. folliculorum also shrank and the stretching frequency of the tails moved faster. When the concentration of the sea mud extract decreased from 2.5 kg/L to 1 kg/L, the proportion of active movement of D. brevis and D. folliculorum decreased from 79.4%（27/34）、65.7%（23/35）to 68.9%（26/38）、53.8%（21/39）, and the activity time extended from 15 min, 20 min to 100 min, 104 min，respectively. The 0.5 kg/L of the sea mud extract showed no significant effect on the morphology and activity of both species of Demodex. When the concentration decreased from 2.5 kg/L to 1.5 kg/L，the time to kill both Demodex spp. extended from 2 h to 8 h. The extract at a concentration of 1 kg/L showed higher acaricidat effect to D. brevis than that of positive control（P＜0.05）, while there was no significant difference for D. folliculorum（P>0.05）. When the concentration decreased to 0.5 kg/L, the acaricidal effect for D. brevis and D. folliculorum remarkably reduced and there was no significant difference with the control groups（P>0.05）. The lowest effective concentration for both Demodex spp. was 1 kg/L. The values of 1 h median lethal concentration（LC50）of the sea mud extract for D. brevis and D. folliculorum were 1 913 mg/L and 2 131 mg/L，respectively.   Conclusion   The water extract of Xiushan sea mud shows acaricidal effect to human Demodex. The effect to D. brevis is slightly better than to D. folliculorum.

Preliminary Study on the Immunological Characteristics of Permissive and Non-permissive Hosts Infected with Schistosoma japonicum

Lu Wei-Yuan, 2, Hu-Yuan, YUAN Zhong-Yang, LI Pei, XU Ye-Shen, CHEN Yu-Juan, ZHOU He-Jun, CHEN Cheng-Xia, CAO Jian-Beng

2011, 29(4):  5-267-271. 

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Objective   To study the difference among immune responses of three kinds of experimental animals with different susceptibility to the infection of Schistosoma japonicum，and preliminarily explore the mechanism of the immune response in permissive and nonpermissive hosts.  Methods   Twelve animals of each kind of rodents，C57BL/6 mice，Sprague Dawley （SD） rats and Microtus fortis，were randomly divided into the infected group and uninfected group each with 6 animals. In infected groups of C57BL/6 mice，SD rats, and M. fortis，each animal was infected with 20，200 and 1 000 cercarie of S. japonicum，respectively. 42 d later，all rodents were sacrificed. Adult worms in portal vein and granulomas in liver were observed and the sera were collected. The levels of cytokines IL-10 and IFN-γ as well as serum IgG，IgG2a, and IgG1 were detected by ELISA .  Results   At the 42th day post infection，worms in portal vein and liver granulomas were observed in C57BL/6 mice and SD rats, but not in M. fortis. The level of IL-10 in the sera of SD rats ［（2.21±0.12） pg/ml］ was significantly higher than that in the sera of M. fortis ［（1.64±0.39） pg/ml］ and C57BL/6 mice［（0.10±0.04） pg/ml］ （P < 0.01）. IL-10 in the sera of M. fortis was also significantly higher than that in the sera of C57BL/6 mice （P<0.01）. IFN-γ in the sera of SD rats ［（0.21±0.11） pg/ml］ was significantly higher than that in the sera of M. fortis ［（0.11±0.03） pg/ml］ and C57BL/6 mice ［（0.09±0.02） pg/ml］ （P<0.05）, but no difference between M. fortis and C57BL/6 mice （P>0.05）. The levels of IgG （1.53±0.31）， IgG1 （1.48±0.44） and IgG2a （0.41±0.11） in SD rats were significantly higher than that in the sera of M. fortis （0.48±0.14， 0.15±0.03 and 0.12±0.06l） （P<0.01）. The levels of IgG （1.21±0.16）， IgG1 （0.88±0.31） in C57BL/6 mice were significantly higher than that in the sera of M. fortis （P<0.01）. IgG1 antibody is the predominant subclass in the three kinds of rodents. The levels of IL-10, IFN-γ and antibody subclass IgG, IgG1, IgG2a in all noninfected rodents were not detected.  Conclusion  IL-10 in nonpermissive hosts，which is an essential agent in the regulation of Th2 immune response，is higher than that in permissive host. It may play an important role in the resistance to schistosome in the nonpermissive hosts.

Effect of Toxoplasma gondii Infection on Cytokines and Spermatogenic Cells in Rats

YANG Rui

2011, 29(4):  6-274-278. 

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Objective   To study on the fluctuation of cytokines of T helper type 1（Th1） and T helper type 2 （Th2） after Toxoplasma gondii infection, as well as pathological damage of testis and apoptosis of spermatogenic cells in male rats.   Methods   Eighty-eight SD male rats (9-10 week old) were randomly and equally divided into normal control group and infection group. Rats in infection group were infected with 1×104 tachyzoites by intraperitoneal injection, while those in normal control group received same volume of PBS. On the day before infection and at the 3rd, 6th, 9th, 12th, ……, and 30th day post infection, four rats from each group were sacrificed for sera and testes. γ-interferon (IFN-γ) and interleukin-4 (IL-4) levels in sera were measured by ELISA. The testes were sliced and observed by microscope. The levels of apoptosis relative proteins Bcl-2 and Bax in seminiferous tubules were detected by immunohistochemistry. Results  IFN-γ in sera of infected rats increased rapidly with the peak ［(518.3±83.6) pg/ml］ at the 6th day post infection, and then decreased rapidly. IL-4 increased slowly with the peak ［(325.0±38.6) pg/ml］ at the 12th day, and then decreased. Both cytokines were significantly higher than the control (P<0.01) in the 30-day period. Pathological examination at the 6th day post infection showed that the cell levels of testicular seminiferous tubule disturbed. The number of primary and secondary spermatocytes decreased significantly. There were few sperms within the lumen or cavity which even closed. All the changes did not recover during 30 days. Bax expression in infected rats significantly increased in spermatogenic cells especially in spermatocytes at the 3rd day (P<0.05), reached a peak （0.547±0.037） at the 6th day, and then gradually decreased to normal after 15 days. The expression of Bcl-2 in infected rats did not change significantly (P>0.05).  Conclusion   T. gondii causes severe pathological damage in spermatogenic cells of the host. During the acute phase there appears Th1 cytokine polarization accompanied by high expression of apoptosis protein Bax, which is mainly expressed in spermatocytes. After rebalance of Th1/Th2, Bax protein expression decreases without noticeable recovery of spermatogenesis.

cDNA Cloning and Sequence Analysis of Musca domestica Antifungal Peptide-1 (MAF-1)

FU Ping, Tun-Jian-Wei, Guo-Guan

2011, 29(4):  7-279-284. 

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Objective   To clone the cDNA sequence of Musca domestica antifungal peptide-1 (MAF-1) and analyze the amino acid sequence of MAF-1 by bioinformatics method.  Methods   Based on the primer designed according to the N-terminal amino acid sequence of MAF-1, the cDNA and amino sequence of MAF-1 were obtained by the methods of RACE and NestPCR. The accuracy of the experiment was confirmed by RT-PCR. The characteristic of the sequence was analyzed by bioinformatics software.   Results   The length of the cDNA sequence of MAF-1 was 568 bp by 3′RACE, including an open reading frame (ORF) of 441 bp length and 3′UTR of 127 bp. It was a novel sequence with the submission number of HM178948 in GenBank since none homology was found when compared with other sequences by Blast. Added with the 9 amino acids that were not used to design primer, the whole sequence of MAF-1 was 156 amino acids conferred from its cDNA. 139 bp cDNA sequence was obtained by 5′RACE and the result was consistent to 3′RACE. The result of RT-PCR showed the cDNA of MAF-1 mature peptide was accurate. The bioinformatics analysis deduced that the theoretic molecular weight and isoelectric point of the whole protein sequence of MAF-1 gene were similar to those detected. The ExPASy illustrated that the MAF-1 gene had a signal peptide. There were abundant α-helix in it, the domain located between the 128 and 153 amino acid residuals. Subcellular analysis showed MAF-1 was almost in the nucleus. PredictProtein found two protein kinase C phosphalation sites and one N-myri-stoylation site, and predicted that it was not a globular protein. In the end, the three dimension image of MAF-1 was set up with 3D-pssm of ExPASy.  Conclusion  The cDNA sequence and the amino acid sequence of MAF-1 have been obtained and analyzed successfully.

Investigation on Anopheles Species and Their Composition in Villages at Different Altitudes of Motuo County，Tibet Autonomous Region

WU Song, SHANG Lin-Hua, ZHOU Shui-Sen, HUANG Fang, HU Guo-Jun, WANG Che-Quan, JIANG Wei-Kang, ZHUO Ma-Yang-Jin

2011, 29(4):  8-285-288. 

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Objective   To study the anopheline species and composition in villages at different altitudes，Muotuo County.  Methods   Six villages with different altitudes were selected as the investigation spots，i.e. Gande，Zhucun，Damu，Motuo，Didong and Beibeng with an altitude 1 966 m，1 510 m，1 408 m，1 178 m，853 m and 831 m，respectively.  Human-baited net traps，cow-baited traps and light traps were set up to collect adult mosquitoes. The trapped mosquitoes were counted and identified according to morphological criteria. Following the classification，the mosquitoes were killed by chloroform and dried on silica-gel，and transported to the laboratory where they were stored at -20 ℃. Species of Anopheles maculatus complex were identified with multiple PCR method.  Results   5 410 anopheline mosquitoes were collected. Two mosquitoes were captured in high altitude village，one was Anopheles gigas bailieyi，while the other was damaged and unable to identify. There were 541（36.9%）An. pseudowillmori，906 An. will-mori（61.7%）and 21 An. peditaeniatus（1.4%）collected in semi-high altitude villages; 260（76.3%）An. pseudowillmori，2 An. willmori（0.6%）and 79 An. peditaeniatus（23.2%）trapped in middle altitude village; and 3 265（90.7%）An. pseudow-illimori，19 An. willmori（0.5%）and 315 An. peditaeniatus（8.8%）trapped in low altitude villages.  Conclusion  An. pseudowillmori，An. willmori and An. peditaeniatus make the main anopheline composition. The proportion of An. willmori is higher than An. pseudowillmori in semi-high alititude villages, while An. pseudowillmori take the absolute predominance in middle and low altitude villages.

Investigation on Angiostrongylus cantonensis Infection in Rodents in Guangdong Province

BO Bei, TUN Jun, RUAN Cai-Wen, LIANG Wen-Jia, DENG Zhuo-Hui, ZHANG Qi-Meng, HUANG Shao-Yu, LIN Rong-Nie, FEI Fu-Quan

2011, 29(4):  9-289-292. 

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Objective   To investigate the natural infection of Angiostrongylus cantonensis in Guangdong Province， and to provide the scientific evidence for control measures.  Methods   The investigation was carried out in 56 villages of 28 towns of 28 counties/districts in East Guangdong， West Guangdong， the mountain area of North Guangdong and Peal River Delta of the Province from 2005 to 2010. The rodents were captured with live trap and the species identified. Angiostrongylus cantonensis adult worms were collected from the hearts and lungs of rodents， examined， counted and the sex of worms identified.   Results   The rodents were captured from 2005 to 2010， belonged to 2 orders， 2 families （sub-family）， 4 genera and 10 species. Seven species of the rodents were found infected with Angiostrongyylus cantonensis in all 28 counties/districts. Totally 5 820 rats were examined and 496 infected ones were identified， with a mean infection rate of 8.52%. The infection rate of rodents was highest in the Peal River Delta， reaching 9.8% （205/2 084） （χ2=15.25， Ｐ＜0.01）. Rattus norvegicus had the highest infection rate of 16.9% （310/1 835）（χ2=240.91， Ｐ＜0.01）. The mean intensity of infection was 6.1 worms/rat. 1 125 female and 1 064 male worms were found respectively（χ2=1.75， Ｐ＞0.05）.  Conclusion   Natural infection of Angiostrongylus cantonensis in rodents has been found in all the 56 villages selected from the 4 regions of Guangdong Province.

Effect of Control Pattern with Emphasis on Canine Deworming in a Pilot of Echinococcosis in Highly Endemic Area，Southern Qinghai Plateau

FU Jing, HAN Xiu-Min, WANG Li-Yang, ZA Xi-Song-Mao, MA Xiao, WANG Yong-Shun, WU Wei-Beng

2011, 29(4):  10-293-295. 

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Objective   To evaluate the effect of control pattern with an emphasis on canine deworming in a pilot of echinococcosis in highly endemic area of southern Qinghai Plateau.  Methods   Four pasturing villages in Xiewu Township of Chengduo County were selected as pilot villages in August of 2008. Baseline survey on awareness of echinococcosis prevention knowledge among residents and status of dogs’infection (coproantigen ELISA) was carried out in the villages. After baseline survey, measures of minimizing the control unit, setting up 15th of each month as fixed canine purgation date(praziquantel 1～2 pill/dog), giving health education to residents, selecting and training control personnel, and mobilizing local residents to participate in control of echinococcosis were performed. In October of 2009, a survey was carried out with the same contents as baseline survey to evaluate the effect of the control pattern.  Results  After intervention，the awareness rate on echinococcosis prevention knowledge in the residents increased from 76.1%（172/226）in 2008 to 98.8%（237/240）（χ2=55.6，P<0.01). The positive rate of coproantigen ELISA for canine echinococcosis decreased from 32.6%（43/132）to 4.2%（5/120）（χ2=32.9，P<0.01).  Conclusion  The  knowledge awareness on echinococcosis prevention in residents increased and infection rate in dogs decreased considerably after intervention.

Efficacy of Compound Dihydroartemisinin/piperaquine in Treatment of Uncomplicated Falciparum Malaria in Myanmar

LIU Hui, Yang-Heng-Lin, Zhang-Jun, Li-Chun-Fu, Nie-Ren-Hua, Wang-Heng-Ye

2011, 29(4):  11-296-298. 

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Objective   To observe the therapeutic efficacy of compound dihydroartemisinin-piperaquine for treatment of uncomplicated falciparum malaria in Myanmar.  Methods  From 2007 to 2008，patients aged 6 to 60 years with uncomplicated P. falciparum infection and parasite density 500 to 200 000 parasites/μl were enrolled following an informed consent. A three-day course of total 8 tablets compound dihydroartemisinin-piperaquine was administered to an adult(each tablet containing 40 mg of dihydroartemisinin and 320 mg of piperaquine phosphate），dosage for children was based on ages（details in the treatment regimen）. The indices including fever subsiding time，parasite clearance time，asexual parasite clearance time and adverse clinical responses were observed and collected on days 7，14，21，and 28 after treatment.  Results  A total of 134 patients completed the treatment. The mean fever subsiding time and mean asexual parasite clearance time were (25.5±2.8）h and （39.5±7.8）h respectively. Asexual parasite clearance rate was 100% on day 7. Four cases recrudesced on day 28 and 16 cases had slight adverse clinical responses such as uncomfortable gastrointestinal tract，headache，nausea，vomit and diarrhea，which disappeared as soon as drug withdrawal.   Conclusion   The compound dihydroartemisinin/piperaquine shows a sound efficacy in treating uncomplicated falciparum malaria.

Clinical Epidemiologic Features in Children Allergic to Dermatophagoides pteronyssinus 

YE Fang, Liang-Jian-Feng, Lou-Jin-Tu

2011, 29(4):  12-299-301. 

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Objective  To investigate the prevalence of Dermatophagoides pteronyssinus in children allergic diseases in Hangzhou and its surrounding areas.  Methods   Western blotting was used to detect the serum antibody for 9 422 children who were admitted due to clinically suspected allergic diseases. Clinical epidemiological features were analyzed for those with total IgE>100 IU/ml and Dermatophagoides pteronyssinus-specific IgE≥0.35 IU/ml.   Results   The prevalence of Dermatophagoides pteronyssinus in children with allergic diseases was 41.2%（3 878/9 422）. The most common symptoms were allergic rhinitis ［47.8%（1 852/3 878）］ and asthma［18.5%（716/3 878）］. The allergic diseases were most prevalent in July, August and October. The diseases were more prevalent in children over 3 years old and above. more in males ［68.8%（2 668/3 878）］ than females［31.2%（1 210/3 878）］.   Conclusion   Data suggest that Dermatophagoldes pteronssinus is an important allergen causing allergic diseases in children in Hangzhou.

Illustrated Keys to Families and Genera of the Superfamily Ixodoidea under New Taxonomic System

CHEN Ze, LI Si-Si, LIU Jing-Ze

2011, 29(4):  13-302-304、309. 

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 Since molecular biology techniques were applied in the phylogeny of ticks in the late 20th century，changes have taken place in the nomenclature and taxonomy of ticks. However， the illustrated keys to families and genera of ticks published in China were incompetence and the taxonomic system was out of date. This article presents a manual of illustrated keys to families and genera of the superfamily Ixodoidea based on the taxonomic system proposed by Barker and Murrell （2004）， and provides a foundation for morphological identification of ticks.

Application of Immune Colloidal Gold Technique on the Diagnosis of Parasitoses

ZHANG Xiao-Xiao, Cui-Li-Yun, Yang-Yi-Mei

2011, 29(4):  14-305-309. 

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Immune colloidal gold(ICG） technique is a simple, rapid, accurate diagnosis method. At present, ICG technique has been widely applied in the accesory diagnosis of parasitoses, and this article reviews the latest progress of ICG technique applied in parasitology.

Research Progress on the Relationship between Osteopontin and Hepatic Echinococcosis

ZHANG Yong-Guo, ZHANG Shi-Jie

2011, 29(4):  15-310-313、318. 

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Osteopontin is a negatively charged， hydrophilic secreted phosphorylated glycoprotein. It is synthesized and secreted by a variety of cells， and is found in various tissues and cells. The protein is similar in structure to matrix proteins， has the characteristics of cytokines in function and invovled in a series of pathological processes. Recent studies confirmed that osteopontin is highly expressed in liver hydatid cyst and may play an important role in cyst formation.

Application of in vitro Cultivation Technique for Metacestodes in Study of Echinococcus spp.

NI Xin-Wei, Jia-wan-Zhong, Zhe-Yong-Hui, Jin-Ke

2011, 29(4):  16-314-318. 

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With the development of the in vitro cultivation of Echinococcus metacestodes，the technique is widely applied in research areas such as the pathogenic biological characteristics and the mechanism of infection and pathopoiesis of echinococcus，and development of novel therapeutic agents against echinococcosis. These will help futher understand the disease and its control. This paper reviews the application of the in vitro cultivation technique of Echinococcus spp.

Retrospective Analysis of 39 Child Cases of Paragonimiasis

GUO Mei, WANG Wei, JIANG Jian-Yu

2011, 29(4):  17-251-253. 

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Clinical data of 39 children with paragonimiasis treated in Chongqing Three Gorges Central Hospital during 2008-2010 were retrospectively analyzed. The cases aged from 3 to 10 years old, with 25 cases of polyserositis （64.1%), 14 cases of cerebral paragonimiasis (35.9%). Among the cases of polyserositis, all showed dyspnea, tachypnea and diminished respiration (100%). Other symptoms or signs included purulent pleurisy, orthopnea, restricted activity, distant heart sounds, purulent pericarditis, abdominal distension, and hepatomegaly. In the 14 cases of cerebral paragonimiasis, 10 cases  (71.4%) complained headache, 8 cases (57.1%) with vomiting, and other symptoms such as seizures, limb rigidity associated with conscious disturbance. Eight patients were treated with surgery and praziquantel, while others with  praziquantel alone. After treatment 25 cases (64.1%) were cured, 13 cases (33.3%) improved, and 1 case (2.6%) showed no change.

Nested PCR for Malaria Detection and Plasmodium Species Identification

SHI Yong-Xia, HUANG Ji-Cheng, SU Jin-Kun, HONG Ye, LI Xiao-Bei, ZHENG Kui, NIE Hu-Qin, GUO Bei-Xuan

2011, 29(4):  18-263-266. 

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According to the sequences of small subunit ribosomal RNA （SSU rRNA） gene of Plasmodium spp.，universal and species-specific primers were designed to detect malaria and identify species. 60 blood samples were detected by the established nested PCR method. The results were compared with those of microscopic examination. 40 blood samples were Plasmodium-positive by nested PCR with 22 samples of P. falciparum，13 of P. vivax，3 with P. falciparum and P. vi-vax mixed infection, 1 of P. ovale and 1 of unclassified malaria infection. Altogether, the coincidence between the results of nested PCR and microscopy stood for 76.7% （46/60），including 18 of P. falciparum，11 of P. vivax and 17 negatives. Further sequence analysis and real-time PCR were performed to detect blood samples with discrepancy，results of which were the same as that of nested PCR. The amplified product of P. ovale was sequenced and showed 100% homology to the corre-sponding part of P. ovale SSU rRNA gene sequence （GenBank No. DQ845247），which confirmed that the case was imported ovale malaria.

The Target of Musca domestica Cecropin on Human Hepatocelluar Carcinoma BEL-7402 Cells

JIN Xiao-Bao, Li-Xiao-Bo, Zhu-Jia-Yong, Lu-Xue-Mei, Shen-Juan, Chu-Fu-Jiang, Mei-Han-Fang

2011, 29(4):  19-271-273. 

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Human hepatocelluar carcinoma BEL-7402 cells were treated with 50 μmol/L Musca domestica cecropin for 12 h, and observed under scanning electron microscope. The effect of Musca domestica cecropin labeled with FITC （FITC-cecropin） on BEL-7402 cells was detected by laser scanning confocal microscopy. The scanning electron microscopy showed that most microvilli on the surface of BEL-7402 cells disappeared at 12 h after cecropin treatment. The laser scanning confocal microscopy revealed that most FITC-cecropin combined with BEL-7402 cell membrane, and partly in the cytoplasm.

Effect of Epitope-based Peptide-DNA Dual Vaccines against Schistosoma japonicum in Mice

WANG Xue-Feng, ZHANG Rong-Bo, HU You-Ying, DU Jiu-Wei, CHEN Xiao-Jun, XU Zhi-Peng, SU Chuan

2011, 29(4):  20-319-320. 

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A C-T-B PDDV mixture of the three constructed epitope-based peptide-DNA dual vaccines（PDDV）containing the CTL （C）, Th （T） and B-cell （B） epitopes from Sj22.6 tegument （C-PDDV, T-PDDV and B-PDDV） with a 1 ∶ 1 ∶ 1 ratio was prepared. Thirty-six mice were randomly divided into six groups averagely named as 18K group, PBS group, C-PDDV group, T-PDDV group, B-PDDV group, and C-T-B PDDV group. All the mice received three immunizations at 2-week intervals with the same dose of antigen（10 μg DNA+28 μg peptide）. One week after the last immunization, the mice were sacrificed, the spleens were removed and splenocytes were collected. Splenocyte proliferation was assayed by［3H］TdR incorporation after stimulation with soluble worm antigen（SWA）. Levels of IFN-r and IL-4 in the splenocyte culture supernatants were determined by ELISA. The results showed that IFN-r content in T-PDDV group ［（76.0±11.2） pg/ml］ was higher than that of PBS ［（13.0±2.1） pg/ml］ and 18K control groups ［（14.0±3.2） pg/ml］ （P<0.01）. IL-4 level in T-PDDV ［（152.0±21.1） pg/ml］ and C-T-B mixture groups［（86.0±12.2） pg/ml］ was higher than others （P<0.01 and P<0.05）. The splenocytes from T-PDDV group showed a significant increase in proliferation compared with PBS and 18K control groups after stimulation by SWA（P<0.01）. However, there was no significant difference in splenocyte proliferation among C-T-B， PBS and 18K control groups （P>0.05）. These findings indicate that T-PDDV and C-T-B PDDV mixture induces stronger immune response than that of C-PDDV or B-PDDV.