Table of Content

30 October 2011, Volume 29 Issue 5

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Construction of GCV-Specific Hammerhead Ribozyme Recombinant Vector of Alpha-8 Giardin in Giardia lamblia

WEI Chao-Jun, LU Si-Qi, CAO Li-Jing, TIAN Xi-Feng

2011, 29(5):  1-321-326. 

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Objective   To construct a GCV-ribozyme recombinant vectors of α-8 giardin in Giardia lamblia.  Methods   The secondary structure of α-8 giardin mRNA （GenBank Accession No. AY781323） was analyzed with the RNA draw software. According to the proportion of G ∶ C and principles of designing hammerhead ribozyme，suitable ribozyme cleavage points were chosen. A specific antisense-hammerhead ribozyme（H8）was designed and synthesized. The ribozyme was cloned into Giardia canis virus （GCV） vector to construct a recombinant viral vector-pGCV634/H8/1423. The vector was linearized and transcripted into the trophozoites of G. lamblia by electroporation method. The α-8 giardin mRNA level of the transfectants and normal trophozoites were analyzed 24 h after electroporation by RT-PCR.   Results   The recombinant vector of GCV-specific hammerhead ribozyme of α-8 giardin in Giardia lamblia（pGCV634/H8/1423） was constructed. RT-PCR assays showed the ribozyme （H8） mRNA can be detected 24 h after transfection and α-8 giardin mRNA was cleaved effectively by ribozyme （H8） introcellularly.   Conclusion   pGCV634/H8/1423 can transfect Giardia trophozoites and cleave mRNA of α-8 giardin intracellularly.

Histopathology Changes in Mice Infected with Toxoplasma gondii Prugniaud Strain

WU Sheng-Wei, BAO Huai-En, LI Xiao-Yan, GE Shuang

2011, 29(5):  2-327-332. 

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Objective   To observe the symptoms and dynamic changes of histopathology in the organs from ICR mice infected by Toxoplasma gondii Prugniaud strain.  Methods   Thirty ICR mice were infected intraperitoneally with cysts, 10 cysts per mouse. 16 mice were injected with PBS. Incidence of the mice was observed. Three mice from the infected group and two mice from the control group were sacrificed, and the liver，spleen, lung, brain, kidney and heart were collected for pathology and immunohistochemistry examinations on the day 5, 10, 15, 20, 25, 30, 60 and 90 post-infection. Results   The infected mice began to fall ill at 6 d post-infection，symptoms including decreased appetite，pilomotor fur，sloth，shakes and diarrhea，with a mortality rate of 20%. From 5 d to 20 d post-infection，microscopic examination for HE stain-slides showed the destroyed liver structure，cellular edema，ballooning change，focal necrosis，sinus hepaticus expansion and hyperemia，and inflammatory infiltration. Splenic corpuscles demolished and disappeared，red pulp widened and white pulp atrophied，splenic sinusoid extended with hyperemia. Lungs showed destruction of the structure and pathological changes of interstitial pneumonia. The pathological changes began to alleviate until recovery after 20 d post-infection. In the brain，neuronal degeneration and necrosis were found at 10 d post-infection. Some neuroglial cell tubercle，blood vessel sleeve cuffing，inflammatory cell infiltration on cavitas subarachnoidealis and cysts were observed from 15 d to 90 d. Granulation tissue was seen at 90 d post-infection. By immunohistochemistry test，internal organs showed toxoplasma antigen at 5 d post-infection，and the positive reaction was remarkable at 10 d post-infection，then began to taper until negative. Toxoplasma antigen was revealed in the brain from 10 d to 90 d post-infection.   Conclusion   Non-specific clinical manifestation and the degeneration，necrosis and inflammatory cell infiltration in poly-organs appear in earlier period of toxoplasma tachyzoite infection in the ICR mice，followed by the co-existing phenomenon of non-specific infection with cysts in the brain.

Impairment of Learning and Memory Ability in Mice with Latent Infection of Toxoplasma gondii

ZHOU Yong-Hua, WANG Xiao-Bo, JIANG Shou-Fu, XU Yong-Liang, TAO Jian-Ping, ZHANG Xiao-Ping, ZHANG Ying, GAO Qi

2011, 29(5):  3-333-338. 

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Objective   To detect the learning and memory ability in mice model of latent Toxoplasma gondii infection with object recognition test and Morris water maze test.  Methods   Thirty-six Kunming mice were divided into control group，infection group with 6 cysts each mouse（low infection group），and infection group with 12 cysts each mouse （high infection group） averagely. Mice in the two infection groups were orally infected with T. gondii Prugniaud （PRU） low virulence strain. Object recognition test was conducted at the 63rd day after infection. After the first day of adaptation and the second day of familiarization in the test，the time expended on exploring new and familiar objects was recorded on the third day and the discrimination index （DI） was calculated. Morris water maze test was conducted at the 66th day. The ability of spatial learning，spatial memory retention and working memory capacity was evaluated by place navigation test，spatial probe test，and working memory test，respectively. The mice were sacrificed at the 74th day after infection. The left cerebral hemisphere of mice was fixed，sliced，and stained with eosin-hema-toxylin for pathological examination. The right hemisphere was used to detect the activity of superoxide dismutase （SOD） and malondialdehyde （MDA） content.  Results  The results of object recognition test showed that the discrimination index of high infection group and low infection group was （14.3±5.2）% and （17.5±5.6）%，respectively，significantly lower than the control ［（28.9±7.1）%］ （P<0.01）. In the place navigation test，the latency to find the platform in the two infection groups was longer than the control，with significant difference on the second and third day （P<0.05）. In the spatial probe test，the percentage of the distance across the platform quadrant in the total swimming distance of high infection group and low infection group were （19.9±5.0）% and （23.9±6.8）%，respectively, significantly lower than the control ［（27.4±3.6）%］ （P<0.05）. In the working memory test，at the fourth day of test the latency of high infection group and low infection group ［（36.5±14.2） s and （35.3±13.7） s］ was significantly longer than the control ［（30.4±12.5） s］ （P<0.05）. In all the tests，there was no statistical significance between low infection group and high infection group （P>0.05）. The brain sections of two infection groups showed cysts of T. gondii，proliferation of glial cells，widened gap around small blood vessels， and a phenomenon of“vascular cuff”. The activity of SOD in the mice brains of two infection groups was significantly lower than the control，while MDA level was significantly higher （P<0.05）. SOD and MDA showed no significant difference between two infection groups （P>0.05）.   Conclusion   Latent infection of T. gondii may lead to learning and memory impairment in mice.

Evaluation of Recombinant SjLAP and SjFBPA in Detecting Antibodies to Schistosoma japonicum

Faustina Halm-Lai, LUO Qing-Li, ZHONG Zheng-Rong, SONG Xiao-Rong, CHEN Zhao-Wu

2011, 29(5):  4-339-347. 

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Objective   To investigate the early response of immunoglobulin G （IgG） antibody responses to Schistosoma japonicum infection in mice by using the recombinant proteins，S. japonicum leucine aminopeptidase （rSjLAP） and S. japonicum fructose-1, 6-bisphosphate aldolase （rSjFBPA）, and evaluate the potential of rSjLAP and rSjFBPA in diagnosis as well as in assessment of therapeutic efficacy in human schistosomiasis.  Methods   rSjLAP or rSjFBPA was induced from Escherichia coli BL21 strain transfected with the expression vectors，pET-28a-rSjFBPA/BL21 or pET-28a-rSjLAP/BL21 using isopropyl-β-D-thiogalactoside （IPTG），and purified by Ni-NTA His Bind resin. 88 BALB/c female mice，inbred and 6 to 8 weeks old，were randomly divided into 4 groups. Groups A，B and C each made up of 21 mice and group D comprised 25 mice. Groups A，B and C were infected with 5，15 and 25 S. japonicum cercariae respectively. As control，mice in group D were left uninfected. 3 mice from each of groups A，B and C were sacrificed and sera collected on days 3，7，10，14，20，30，and 60 post infection. All the 25 mice in group D were sacrificed on the first day of the experiment for serum collection. rSjLAP and rSjFBPA were screened and used in ELISA to test the antibody response of the serum samples. Also，sera of 38 acute patients，96 chronic patients with schistosomiasis japonica，90 healthy donors and patients with other parasite infections including Clonorchis sinensis （33 cases），Paragonimus westermani （40） and hookworms （37） were tested using the recombinant protein-based ELISA. In addition，36 sera each from the acute and chronic patients 12 months after treatment with praziquantel and 64 of the chronic patients in more than 2 years post-treatment of praziquantel were tested. The dosage of praziquantel for both acute and chronic patients was 60 mg/kg，2 times/d×2 d.  Results   IgG antibody response was first detected at day 10 post infection by rSjLAP，rSjFBPA or the combined antigen assay. The mean absorbance （A450） on this day were 0.535±0.053，0.595±0.033，0.696±0.104 for group B;  0.548±0.060，0.608±0.063，0.621±0.090 for group C; and 0.415±0.038，0.455±0.056，0.498±0.077 for group A for rSjLAP，rSjFBPA and the combined assay respectively （P<0.05）. Early antibody level to both antigens was significantly higher in mice infected with 15 or 25 cercariae than those with 5 cercariae （P<0.05）. However，ELISA results in patients with confirmed schistosomiasis revealed positive rates of 97.4% （37/38） and 87.5% （84/96） for acute and chronic schistosomiasis with rSjLAP ，94.7% （36/38） and 88.5% （85/96） for acute and chronic schistosomiasis with rSjFBPA and 94.7% （36/38） and 85.4%（82/96） with both rSjLAP and rSjFBPA respectively. Statistical analysis showed no significant difference in the positive rate （P＞0.05）. Also，rSjLAP and combined antigens showed a specificity of 96.7% （87/90） while that of rSjFBPA was 97.8% （88/90）. There was a general decrease in the antibody titer of the patients after treatment. In 12 months after treatment it was 0.236±0.212 with rSjLAP，0.287±0.191 with rSjFBPA，and 0.235±0.120 with both antigens respectively for acute cases；For chronic patients, it was 0.266±0.124，0.261±0.143 and 0.265±0.140 in 12 months post-treatment, and 0.204±0.074，0.176±0.074，and 0.176±0.073 in 2 years, respectively. For healthy control, it was 0.188±0.056, 0.173±0.45， and 0.184±0.051, respectively. No significant difference on antibody titer was found between treated patients and control （P>0.05）. The cross reaction with C. sinensis was 15.2%(5/33） for rSjLAP，12.1% （4/33） for rSjFBPAand 9.2% （3/33） for combined antigens. With P. westermani, it was 15.0% （6/40），12.5% （5/40） and 15.0% （6/40）, respectively, and 8.1% （3/37） with hookworm infection.  Conclusion  The study showed a satisfactory sensitivity and specificity of rSjLAP and rSjFBPA by ELISA which is promising for the immunological diagnosis of schistosomiasis.

Investigation on the Hosts with Natural Paragonimus Infection and Species Identification in Jinhua Prefecture of Zhejiang Province

LOU Hong-Jiang, HU Ye, JIN Yao-Jian, TU Xin-Tu, WANG Lan, HE Xu-Ying, TU Ping-Guang

2011, 29(5):  5-348-352. 

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Objective   To investigate the natural hosts infected with Paragonimus sp. and identify the species of the parasite in selected counties/districts of Jinhua prefecture in Zhejiang Province.  Methods   Three townships/towns were randomly sampled from each of the 9 counties/districts in Jinhua as pilot spots for the survey. Fresh-water snails were collected from the fields for examining cercariae. Crabs were collected and detected for metacercariae by routine technique and the metacercariae were fed to dogs purchased in areas free from paragonimiasis. Fecal materials of dogs and cats around the villages and streams where crabs were found infected were collected for examining eggs. The artificially infected dogs were sacrificed 55 d after infection to receive adult worms. The size of cercariae, metacercariae, eggs and adult worms was measured. After the DNA of the adult worm was extracted，PCR was used to amplify the COI gene and ITS2 gene of the mitochondria from the worms. Homology with relative strains/isolates was analyzed and phylo-genetic tree constructed.  Results   The survey demonstrated that the snail Semisulcospira libertina and the crab Sinopotamon chekiangense served as the first and second intermediate hosts respectively. Natural infection was found in Wucheng District with an infection rate of 0.2% (2/1 088)in snails and 76.7%(46/60)in crabs in Shafan township, and an infection index(II)of 2.0 in crabs, 0.1% (1/1 683)in snails and 53.0%(46/60)in crabs with an II of 0.9 in Langya town. The infection rate was 0(0/575)in snails and 30.0%(18/60)in crabs with an II of 0.1 in Baimu township of Wuyi County. Paragonimus eggs were detected in feces of stray cats with a positive rate of 8.3%(1/12)in Shafan and 0.6%(1/17)in Langya. The size and morphology of the cercariae, metacercariae, eggs and adult worms were similar to those of Paragonimus westermani. The sequences of the COI and ITS2 genes were with 390 bp and 363 bp respectively, indicating a homology of 88.2%-98.2% and 86.5%-88.1% to the 11 strains/isolates of P. westermani recorded in the GenBank. The phylogenetic trees revealed that the parasite in Jinhua was in between the Minchin(Minqing, Fujian)strain and the Japanese Mie and Chiba strains.  Conclusion  Natural paragonimus infection has been detected in snails, crabs and cats in Jinhua. The parasite has been identified as P. westermani which is phylogenetically close to those reported from Fujian Province and from Mie and Chiba of Japan.

Experimental Treatment on Alveolar Hydatid Disease in Mice with Multi-frequency Focused Ultrasound

YE Hua, WANG Wei, JING tao, HAN Jian, BAO Gen-Shu, FAN Lin-Lan, LIANG Xiao-Ping, LU Jun

2011, 29(5):  6-353-357. 

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Objective   To investigate the efficacy of multi-frequency focused ultrasound （MfFU） in the treatment of alveolar hydatid disease in mice.  Methods   Thirty Kunming mice infected subcutaneously with alveolar protoscoleces were divided into 3 groups randomly of 10 mice each and irradiated with different intensity of MfFU. Mice in the experiment groups B and C were irradiated only once for 5 min and group A served as control. The irradiation power of the 3 transducers in the low-power group （group B） was 4 W+4 W+5 W；that in the high-power group （group C） was 10 W+11 W+10 W. After the irradiation，the morphological change of alveolar tissues was observed with transmission electron microscope. The survival rate of protoscoleces was evaluated with methylene blue staining. The mitochondrial content in the alveolar tissues was detected with laser confocal microscope. The Coomassie brilliant blue staining and succinate dehydrogenase （SDH） kit were applied for measuring the amount of general proteins and the activity of succinate dehydrogenase of the alveolar hydatid cyst respectively.  Results   After irradiated by MfFU，transmission electron microscopy showed that in groups B and C the cells on the germinal layer decreased. The mitochondria swelled or broke. The endocytoplasmic reticulum became swollen markedly. The karyotheca looked unclear. The microvilli shortened or disappeared. All the damages in group C displayed more seriously than in group B. The survival rate of protoscoleces in groups B （70.50%） and C （59.83%） was statistically lower than that of group A（82.33%） （P<0.05）. And there was also a statistical difference between groups B and C （P<0.05）. The amount of general proteins and the activity of SDH in groups B （3.07 mg/ml and 2.15 U/mg respectively） and C （2.87 mg/ml and 1.87 U/mg） were lower significantly than those in group A （3.83 mg/ml and 3.50 U/mg）（P<0.05），but no statistical difference between B and C （P>0.05）. The mitochondria content in groups B（105.46 a.u/a） and C（70.90 a.u/a） was lower than that in group A （133.45 a.u/a）. Group C showed statistical difference than A and B （P<0.05），but there was no statistical difference between A and B（P>0.05）.  Conclusion It is evident that the cysts of Echinococcus multilocularis in mice can be damaged by MfFU which shows certain curative effect.

Detection and Identification for Gnathostoma sp.in Imported Monopterus albus

LI Shu-Qing, LI Wen-Wen, CHEN Zhi-Fei, LI Jian, CHEN Shao-Hong, ZHANG Yong-Nian, HUANG Wei-Xi, WANG Qiao-Quan

2011, 29(5):  7-358-362. 

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Objective   To inspect the third stage larvae of Gnathostoma in imported Monopterus albus，and identify its species.  Methods   Ten batches of M. albus imported to Shanghai were detected for nematode Gnathostoma from January 2010 to March 2011. Fifty-two M. albus imported from the Philippines （25）， Indonesia （24） and Bangladesh （3） were sampled （3-10/batch），which were dissected，minced，and digested. The suspension was filtered with 10 mesh screen to take the disposit. The complete parasites were picked out under stereoscope followed by morphological identification. The rate and intensity of infection were calculated. Genomic DNA of Gnathostoma was extracted to amplify internal transcribed spacer region 2 （ITS-2） and cytochrome C oxidase subunit 1 （cox1） by PCR，the product of which was analyzed by electrophoresis and sequencing. The sequences were aligned with corresponding sequences in GenBank.  Results  The third stage larvae of Gnathostoma were detected in M. albus from Indonesia and Philippines with infection rate of 36.0% （9/25） and 50.0% （12/24） and average infectiosity of 7.8 （70/9） and 2.8 （34/12），respectively. No Gnathostoma was found in M. albus imported from Bangladesh. Under microscope，the larvae showed one cephalic bulb with 4 rings of hooklets on it，cross striations and small spines on the body surface. The front body spines were bigger and denser，while the rear spines were smaller and sparser. It had 1 cervical papilla and 4 cervical capsules. Morpho-logical characteristics were similar to the third stage larvae of G. spinigerum. PCR results showed that the length of the ITS-2 and cox1 PCR products was 647 bp and 441 bp，respectively. Sequence alignment analysis showed that the two PCR products had 99%-100% consistency with G. spinigerum ITS-2 （GenBank Accession No. AB181155 and Z97175） and cox1 （GenBank Accession No. AY501388，AB180099, and AB551552）.   Conclusion  All the larvae detected in M. albus imported from the Philippines and Indonesia have been identified as G. spinigerum.

Protein Interaction between Aldolase and Actin of Toxoplasma gondii by GST Pull-down

ZHENG Bin, YIN Zhi-Kui, HE Ai, LI Zhuo-Ya, ZHAN Xi-Mei

2011, 29(5):  8-363-367. 

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Objective   To identify the protein-protein interaction between aldolase and actin of Toxoplasma gondii by GST pull-down.  Methods   The aldolase and actin genes were obtained from cDNA library by PCR amplification，and subcloned respectively into pGEX-4T-1 and pET30a. The fusion protein GST-Aldolase and Actin-His6 were expressed in E. coli upon induction by 1 mmol/L IPTG and then purified with affinity chromatography. Fifteen rats were immunized intradermally with 200 μg Actin-His6 protein per rat at first time to produce the polyclonal antibodies. Then 100 μg Actin-His6 protein per rat on the 2nd-4th immunizations. Rats were immunized for 4 times with 7 days interval. The serum of rats was collected from heart at the fifth day after the final immunization. Glutathione sepharose beads were incubated with GST-Aldolase protein，then incubated with Actin-His6，and bound proteins were eluted using sample buffer. Eluants were resolved by SDS-PAGE and Western blotting.  Results   The aldolase and actin genes were obtained, and the recombinant plasmid aldolase/pGEX-4T-1，actin/pET30a were successfully constructed. Protein GST-Aldolase and Actin-His6 were expressed and purified in vitro. Serum samples were prepared from rats immunized with protein Actin-His6, and polyclonal antibody was purified with affinity chromatography. SDS-PAGE and Western blotting analysis of products from GST pull-down experiment showed that the protein bands on NC membrane were specifically recognized by anti-Aldolase-His6 and anti-Actin-His6 antibody.   Conclusion  Aldolase interacts with Actin of Toxoplasma gondii.

Effect of Physicochemical Factors on Infectivity of Spirometra mansoni Plerocercoid

TANG Gui-Wen, CHEN Yan

2011, 29(5):  9-368-371. 

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Objective  To observe the effect of different physicochemical factors on the infectivity of Spirometra mansoni plerocercoids.  Methods  The muscle samples with plerocercoids taken from Rana nigromaculata （about 1 cm³ each piece） were treated with different temperature （－20 ℃, 4 ℃, 37 ℃ and 56 ℃） or different concentrations of ethanol （20%, 30%, 40%, 50% and 60%） for 1, 2 or 3 h, or soaked in ginger juice, vinegar （total acid concentration of 4.5%, pH 3.05） or soy sauce （containing 19.3% NaCl） for 3, 6, 12 or 24 h. The muscle with plerocercoids treated with normal saline under 20 ℃ served as control. 30 plerocercoids were used under each condition and fed to 10 mice averagely (3 larvae/mouse). Another 20 plerocercoids with frog meat were comminuted for 3 min then fed to 10 mice. One week later, the mice were sacrificed to collect the parasitic plerocercoids and the number of positive mice and plerocercoids was recorded.  Results   None of the mice fed with plerocercoids treated under －20 ℃ for 2 h was infected. All the mice fed with plerocercoids treated under 56 ℃ for 2 h and 3 h were infected. The percentage of infective plerocercoids was 60% (18/30) and 43% (13/30)，respectively，considerably lower than those of the control （90%，27/30） （P<0.05）. None of the mice fed with plerocercoids soaked in 60% ethanol for 2 h was infected. All the mice fed with plerocercoids soaked in 60% ethanol for 1 h, or in 50% ethanol for 2 h or 3 h were infected. The percentage of infective plerocercoids was 60% (18/30), 57%(17/30)，and 50%(15/30), respectively，considerably lower than those of the control （93%，28/30） （P<0.05）. None of the mice fed with plerocercoids soaked in vinegar for 24 h，or soy sauce for 6 h was infected. The infectivity of the plerocercoids treated by ginger juice for 24 h was similar to the control（P>0.05）. Among the ten mice fed with comminuted frog meat with plerocercoids, five were infected with 6 plerocercoids.   Conclusion  Treatment with －20 ℃ or 60% ethanol for 2 h， soy sauce for 6 h，or vinegar for 24 h can destroy the infectivity of plerocercoids in 1 cm3 frog muscle.

Therapeutic Efficacy and Safety of Compound Dihydroartemisinin/pipera-quine for Uncomplicated Plasmodium falciparum Infection in Laiza City of Myanmar Bordering on China

SUN Xiao-Dong, ZHANG Zai-xin, WANG Jian, DENG Yan, YANG Yuan-Chuan, Lasi Ja-hkawn, SUN Xiao-Yang, WANG Heng

2011, 29(5):  10-372-375. 

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Objective   To assess the efficacy and safety of compound dihydroartemisinin/piperaquine（DHAPIP） for treating uncomplicated falciparum malaria in Laiza city of Myanmar at the China-Myanmar border area.  Methods   A clinical trial was conducted in Laiza City and its four suburban natural villages bordering with China from September to December in 2008. Patients（aged 2-60 years）infected by Plasmodium falciparum without clinical complications  （≥250 asexual parasite·μl-1 of whole blood）were recruited for the assessment. The cases were given a 2-day course with DHAPIP tablets each containing 40 mg of dihydroartemisinin and 320 mg of piperaquine phosphate, and the total dosage varied with the body weight. For example, a patient with 50 kg body weight was given 8 tablets divided into 4 times at an interval of 8-10 h. The cases were then followed-up at D0，D1，D2，D3，D7，D14，D21 and D28 for observing their symptoms, the density of parasite, body temperature and side reaction. The therapeutic efficacy was assessed by using WHO classification of therapeutic response to the treatment of antimalarial drugs, including the time of fever subsidence, the clearance time of asexual parasites and the clearance rate of gametocytes.  Results   Among the 74 cases enrolled, 64 completed 28-day follow-up. The therapeutic efficacy reached 100% with adequate clinical and parasito-logical responses. The mean fever subsidence time was（22.5±8.2）h. The median of clearance time of asexual parasites in blood was 30.0 h[（17.1～168.2） h]. The rate of eliminating asexual parasites and fever subsidence in D3 and D7 was（93.8% and 100%）and（100% and 100%），respectively. The clearance rate of gametocytes in day-28 was 75.0％. It showed 9.9% of side reaction with 7 cases suffering from mild adverse responses among 71 of full-course medication.  Conclusion  DHAPIP is efficacious and safe for the treatment of uncomplicated falciparum malaria in Laiza city of Myanmar in the border area.

The Impact of Toxoplasma gondii Infection on Host Cell Signal Transduction

LUO Ji-Xuan, ZHU Guo-Hui, WU Yong-Jian, PENG Hong-Juan

2011, 29(5):  11-378-384. 

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In the process of invasion and development in host cells，Toxoplasma gondii causes acute and chronic infection. The parasite manipulates the host cell elaborately and integrately to keep a delicate balance between induction and elimanation of the host cell immune reaction. It can then dwell and multiply successfully in the host cell，and hopefully be transmitted to a definitive host. The host cell signaling is changed and regulated extensively by the parasite in the process，which plays vital roles in parasite invasion and development. This review shed light on the manipulation of host cell signaling by T. gondii infection in these aspects：① T. gondii secreted proteins which manipulate host cell signaling； ② T. gondii modulates the innate and protective immune related host cell signaling；③ T. gondii regulates anti-apoptotic reaction and cell cycle related host cell signaling；④ T. gondii adjusts calcium relevant host cell signaling；⑤ T. gondii manipulates cell structure reorganization relevant host cell signaling.

Application of Molecular Biological Techniques in Taenia Identification

LI Yan, LIU Hang, YANG Yi-Mei

2011, 29(5):  12-385-388. 

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The traditional identification of Taenia spp. based on morphological features of adult and cysticercus has difficulties in identifying the morphologically similar species. The recent development of molecular techniques provides more scientific ways for distinguishing Taenia species. This paper summarizes the application of molecular biological techniques in the identification of Taenia, such as analysis of DNA sequence, PCR-RFLP and LAMP.

Application of Toll-like Receptor Agonists as an Adjuvant Component to Malaria Blood-stage Vaccine

XIE Chuang-Bo, QIAN Feng, XU Lu-Ji

2011, 29(5):  13-389-393. 

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Toll-like receptors （TLRs） belong to the category of pattern recognition receptor. The binding of TLRs with their respective ligands activates innate immune system，thereby initiates adaptive immune responses. As such，some TLR ligands or agonists have been used as an adjuvant component in a variety of vaccine formulations. AMA1 and MSP1 from Plasmodium falciparum are two main antigens of malaria blood-stage vaccine，but they are poor immunogens in humans. To enhance the immunogenicities of these two vaccine candidates，the TLR agonists have been used in their formulations for the clinical trials. Recent progress in the field is reviewed in this article.

The Student Submission Ways of Experiment Report Based on Digital Microscopic System in Parasitology Teaching

LIANG Yu-Fen, Chen-Hai-Ying, Hui-Jun-Bin

2011, 29(5):  14-397-398. 

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When the digital microscopy interactive system is applied to parasitology experiment teaching，students can submit their experiment reports in two ways：a paper document on a drawing paper or an electronic document taken images with the system. Submission of a paper report needs more time but requires the students to work more carefully，and an electromic document allows them to have more time to observe the specimen and work in a higher efficiency. It would be better to ask students to do both.

Changes of Superoxide Dismutase and Lipid Peroxide in Rats with Pneumocystis carinii Pneumonia

LIU Xin-Hui, ZHOU Bi-Ying, DAI Xiao-Huang

2011, 29(5):  15-375-377. 

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Sprague-Dawley（SD） rat model of Pneumocystis carinii  pneumonia（PCP） was established by groin subcuta-neous injection with dexamethasone sodium phosphate 3.5 mg each twice a week for eight weeks. There were two groups：infected group（eighteen rats）  and normal control group（six rats）. Pathological changes in lung tissues were observed in the lung imprint after staining with Gomori methenamine silver（GMS）and in tissue sections after staining with hematoxylineosin（HE）. The activity of superoxide dismutase（SOD）and the content of lipidperoxide（LPO）in lung tissue homogenate were detected by spectrophotometric method. Results showed that in infected group more PC cysts were found in the lung imprint and typical pathological change observed in the lung section. SOD acitvity in infected group[（31.49±7.18） U/mgprot]decreased significantly compared with the control[（54.41±8.97） U/mgprot]（P<0.01），but LPO in infected group[（2.26±0.21） nmol/mgprot]was higher significantly than the control[（1.63±0.01） nmol/mgprot]（P<0.01）.

Research on the Area Distribution and Host Selection of Leptotrombidium scutellare in 19 Counties of Yunnan Province

DAN Yin-Zhu, GUO Xian-Guo, ZUO Xiao-Hua, WANG qiao-Hua, WU Dian

2011, 29(5):  16-393-396. 

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In order to investigate the area distribution and host selection of Leptotrombidium scutellare in Yunnan Province，a field survey was carried out during 2001 to 2009，based on different geographic location，topography，climate and ecological characteristics. A total of 16 491 L. scutellare were captured from the body surface of 9 838 small mammal hosts of 7 families，18 genera，and 30 species in 4 orders，accounted for 17.73%（16 491/92 990） of all chigger mites collected. L. scutellare distributed in 12 counties，more in the northwest and south of the Province. Although L. scutellare could parasitize on different small mammal species，most of them were on Eothenomys miletus and Apodemus chevrieri.

An Easy Way to Purify the Inclusion Body Protein with High Purity from Prokaryotic Expression Cells

LIU Rong, Zhong-xin-Ping, Jiang-Ming-Sen, Dong-Hui-Fen

2011, 29(5):  17-399-封三. 

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To clone partial ORF of SjBMP and to construct the recombinant SjBMP-pET-28a(+） plasmids, and then to transform them into the competent cells E. coli BL21 (DE3）, finally a positive clone was used to be induced by IPTG. The bacterial aggregates with target protein expressed as inclusion bodies were purified by the methods of Ni²⁺-NTA affinity purification under denaturation condition and SDS-PAGE gel extraction. The purified protein was used to immune rabbits and make antiserum against the SjBMP, and  the antiserum were then used to identify the rSjBMP by Western blotting. The target protein obtained by Ni²⁺-NTA  Agarose affinity purification was not pure with unspecific proteins, but the protein further purified by SDS-PAGE gel extraction and the dialysis bag horizontal electrophoresis was quite pure, and the recovery rate was more than 11.0%. Meanwhile, Western blotting was used to identify the recombinant SjBMP protein by antiserum, only a specific single strip appeared, which suggested the protein purified by this method kept its anti-genicity, and could be used for common immunological studies. Therefore, the SDS-PAGE gel extraction combining with electroosmosis and dialysis recycling are good and easy to purify the inclusion body proteins.