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    28 February 2015, Volume 33 Issue 1
    Analysis of Malaria Epidemic Characteristics in Anhui Province#br#  during 1999-2013
    XU Xian,LI Wei-dong*,JIANG Jing-jing,ZHANG Tao,WANG Jian-jun
    2015, 33(1):  1-1-6. 
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    Objective  To analyze the characters of malaria epidemic situation in Anhui Province from 1999 to 2013.  Methods  The incidence and epidemiological data of malaria were retrospectively analyzed by Microsoft Office Excel 2007 and MapInfo Professional 7.0 software.  Results  The number of malaria cases changed significantly from 1999 to 2013. In the growing period(1999-2006), the annual malaria incidence increased from 1.32/100 000 to 57.16/100 000. The turning point was in 2007 with an annual incidence of 44.69/100 000. From 2008 to 2013 (the reducing period), the annual incidence declined from 22.04/100 000 to 0.32/100 000. All the indigenous cases were vivax malaria. The number of indigenous cases increased from 814 in 1999 to 34 982 in 2006, and then reduced to 3 in 2013, since 2007 the comprehensive measures with an emphasis on the clearance of malaria infection source were implemented. The cases were mainly distributed in the area along the Huaihe River and its north. The epidemic areas moved from the middle to the northern part of the province in the growing period. The incidence in the northern part was falling faster than the middle area in the reducing period. More cases occurred in summer and autumn. Since 2009 the number of imported cases increased from 18 in 2009 to 191 in 2013. During 1999-2010 all the imported cases were falciparum malaria. Since 2011 the Plasmodium parasites were more diverse. The imported cases mainly occurred in the counties with more exported laborers.  Conclusion  The epidemic of indigenous malaria has been effectively controlled in Anhui Province, while the imported cases have increased in recent years.

    Application of Semi-solid Culture and Rapid Screening Strategy in the Preparation of Monoclonal Antibody against Plasmodium falciparum Histidine-rich Protein
    JIANG Li*,MA Xiao-jiang,ZHANG Yao-guang,JIANG Shou-fu
    2015, 33(1):  2-7-13. 
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    Objective  To prepare the monoclonal antibody of Plasmodium falciparum histidine-rich protein, and analyze the roles of semi-solid culture technique and screening strategies in the preparation of monoclonal antibodies.  Methods  BALB/c mice was immunized with the recombinant antigen of Plasmodium falciparum histidine-rich protein(rePf-HRP). The spleen cells of immunized mice were fused with SP20 cells. The fused cells were cultured in semi-solid medium containing 2% methylcellulose to promote colony growth. The single cell clone was transferred to a liquid medium. Testing methods for screening culture supernatant was established based on the immune antigen and other related antigens. The positive cell clones by coarse screening and specific screening were preserved in liquid nitrogen. The positive cell lines were used for ascite antibody preparation, identification of IgG subclass, recognition sites, antibody affinity and application analysis on sensitivity of detecting antigen.  Results  A total of 915 cell clones were obtained in semi-solid culture after cell fusion. The positive rate by coarse screening was 37.8% (346/915). The positive rate of specific screening accounted for only 2.6% (9/346) of the coarse screening-positive clones, 0.98% (9/915) of the total number of clones. Eight specific antibody-secreting cell clones were obtained after liquid nitrogen frozen recovery tests. After further detection, 2 specific cell clones could be used as a pair of antibody for rapid detecting circulating antigen in the blood of patients with falciparum malaria. Conclusion  Semi-solid culture method can provide enough fused cells for screening. Combined strategy of coarse and specific screening ensures the rapid selection of specific clones.

    Construction and Identification of Transgenic Strain of Toxoplasma gondii High-expressing Virulence Factor ROP18
    JIANG Zong-ru1,AN Ran1,YANG Jie2,WAN Li-juan1,DU Jian1,3 *
    2015, 33(1):  3-14-18. 
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    Objective  To construct a transgenic strain of Toxoplasma gondii high-expressing ROP18.  Methods The gene sequence of encoding ROP18 was amplified by RT-PCR with RNA of T. gondii RH strain. The purified PCR product was subcloned into pCR-Blunt Ⅱ-Top vector to construct pROP18. The gene sequence of encoding ROP18 was amplified from pROP18, and subcloned into pTUB8-mycGFPPftail-Ty1. The recombinant plasmid pTUB8-ROP18-Ty1 was electroporated into T. gondii RH strain. Stable transgenic cells were selected in the presence of 25 μg/ml mycophenolic acid and 50 μg/ml xanthine, and parasite clones were isolated by limiting dilution after drug selection. The expression of ROP18 in transgenic parasites was detected by immunofluorescence analysis and Western blotting. Giemsa assay was used to detect the proliferation rate of RH strain and the transgenic strain of T. gondii high-expressing ROP18. Twenty mice were divided into two groups. Each mouse in RH strain group was intraperitoneally injected with 1×103 RH strain, and that of transgenic strain group received 1×103 transgenic high-expressing ROP18 strain.  Results  The full-length sequence of ROP18 gene(1 665 bp) was amplified by RT-PCR. The recombinant plasmid pTUB8-ROP18-Ty1 was identified by restriction enzyme digestion and sequencing methods. Western blotting analysis showed that the transgenic high-expressing ROP18 strain expressed ROP18-Ty1(Mr 56 000). Immunofluorescence assay showed that ROP18-Ty1 was localized in the rhoptry of T. gondii. Giemsa assay confirmed that ROP18 protein enhanced the proliferation of T. gondii. On the 6th, 12th, and 24th hour after HFF cells infected with T. gondii, the number of tachyzoites in transgenic strain group was 100.0±16.9, 476.0±31.1, and 860.0±52.3, respectively, higher than that of RH strain group (88.0±16.9, 300.0±11.3, 675.0±35.4)(P<0.05). In RH strain group, all the mice were survival on the 8th day post-infection, the survival rate on the 14th day was 30% (3/10), and all died on the 16th day. In transgenic strain group, all the mice were survival on the 5th day post-infection, the survival rate on the 8th day was 30%(3/10), and all died on the 9th day.  Conclusion  The transgenic high-expressing ROP18 strain of T. gondii is constructed.

    Prokaryotic Expression and Immunoreactivity Analysis on Profilin of Toxoplasma gondii
    YUAN Fei1,LIU Zhuan-zhuan2,ZHANG Bo2,CAO Jian-ping3,ZHENG Kui-yang2 *,WANG De-guang1
    2015, 33(1):  4-21-24. 
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    Objective  To clone and express the profilin(PRF) gene of Toxoplasma gondii, and analyze the immunoreactivity.  Methods  Total RNA was extracted from tachyzoites of T. gondii RH strain. The coding region of TgPRF was amplified with a pair of specific primers. PCR product was digested with double restriction enzyme and ligated into pET30a(+) vector. The recombinant pET30a(+)-TgPRF plasmid was transformed into E. coli DH5α with positive clones confirmed by the double restriction enzyme digestion, PCR and sequencing. The correct plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed proteins were purified with Ni-NTA affinity chromatography and analyzed by SDS-PAGE. Western blotting with rabbit anti-T. gondii serum was used to analyze its antigenicity.  Results  The product of RT-PCR was with 492 bp. pET30a-TgPRF was confirmed by the double restriction enzyme digestion, PCR and sequencing. SDS-PAGE analysis showed that the expressed product was a soluble protein with a relative molecular weight of 35 000. Western blotting assay revealed that rTgPRF was recognized by rabbit anti-T. gondii serum.  Conclusion  TgPRF gene has been expressed in prokaryotic expression system and shows immunoreactivity.

    Overexpression of Toxoplasam gondii ROP18 by Tet-on Lentivirus Expression System
    WANG Xiao1,ZHU Jun1,WU Liang1,WU La-mei1,LIU Yuan1,SU Dan-hua1,DING Ning1,ZHAO Kang-rong1,JIANG Xu-gan1,CHEN Sheng-xia1 *,CAO Jian-pin2
    2015, 33(1):  5-25-28. 
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    Objective  To construct a 293T mutant cell line over-expressing ROP18 of Toxoplasma gondii by Tet-on lentivirus expression system.  Methods  Rop18 gene of T. gondii was amplified by PCR, and inserted into a lentiviral vector pLVCT-tTR-KRAB. The recombinant plasmid pLVCT-tTR-KRAB-ROP18(6 μg) and 293T human embryonic kidney cells were co-transfected with psPAX2 (4 μg) and pMD2.G (2 μg) for the packaging. The result of co-transfection was evaluated by fluorescence microscopy. At 48 h and 72 h after co-transfection, the supernatant of the packaging lentivirus was collected for the 293T cell infection. The doxycycline(DOX) was added into the medium to induce the ROP18 expression in 293T cells. The ROP18 fusion expression was observed under fluorescence microscope and detected by RT-PCR after induction.  Results  PCR product of the gene fragment encoding ROP18 was 960 bp. The recombinant plasmid pLVCT-tTR-KRAB-ROP18 was identified by PCR and restriction enzyme digestion. Green fluorescence was observed in 293T cells at 48 h post-transfection. Bright green fluorescence was observed in 293T cells at 24 h after DOX induction. RT-PCR results showed that a 960 bp specific band(ROP18 gene) was detectable in 293T cells.  Conclusion  293T cell line stably expressing ROP18 is established with Tet-on lentivirus expression system.

    Effect of Toxoplasma gondii Infection in Female Mice on Dopamine Level in the Brain of the Male Offspring
    ZHOU Yong-hua1,2,ZHU Hu-ping3,XU Jin-jun2,ZHANG Ying1,MAO Ai-min3,YANG Jing1,FAN Feng3,XU Yong-liang1,ZHAO Zhong-xing3,TAO Jian-ping2 *
    2015, 33(1):  6-29-32. 
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    Objective  To investigate the effect of Toxoplasma gondii infection in female mice on dopamine level in the brain of male offspring.  Methods  Thirty-six ICR female mice were randomly divided into control group and infection group, 18 mice in each group. Each mouse in infection group was orally infected with 10 cysts of T. gondii Prugniaud strain. On the 90th day after infection, the infected female mice were mated with normal male ICR mice at 1 ∶ 1 ratio. On the 20th day of pregnancy, 2 mice in each group were delivered for fetal mice by cesarean section, and the brain of male fetal mice (n=6) in each group were collected. On the 14th and 63rd day after birth, 6 male offspring mice in each group were sacrificed, and the brain were collected. Dopamine levels in the cortex, cerebellum, hippocampus, and striatum were analyzed by high-performance liquid chromatography-electrochemical detection(HPLC-ECD).  Results  Three mice in infection group died during the experiment, and 6 out of 15 female mice mated successfully. The number of fetal mice and F1 generation mice in infection group was 12 (male: 7) and 21 (male: 15), respectively. All the mice in control group mated successfully. The number of fetal mice and F1 generation mice was 23(male: 12) and 179(male: 92), respectively. The dopamine level in the cerebellum of fetal mice of infection group and control group was (413.25±21.78) ng/g and (346.30±51.83) ng/g, respectively(P<0.01). No significant difference was found in dopamine content in the cortex between the two groups (P>0.05). Compared with the control  group, on the 14th day and 63rd day after birth, the dopamine content in cortical areas[(462.50±24.80) ng/g and (1 215.77±113.64) ng/g], cerebellum area[(271.55±26.19) ng/g and(1 328.82±39.62) ng/g], hippocampus area [(225.78±24.17) ng/g and (1 322.70±58.34) ng/g], and striatum area [(455.23±61.53) ng/g and (991.32±54.31) ng/g] of the male offspring in infection group were significantly higher than that of the control (P<0.05, P<0.01).  Conclusion  T. gondii infection in female mice causes an increase of dopamine level in the brain of F1 generation male mice.

    Species Identification of Puparium Samples of Common Sarcosaphagous Flies Based on Molecular Marker Analysis
    LI Xue-bo1,NING Shu-hua2,ZHANG Gui-qin3,YU Hong-li3,WANG Qing-shan4,Li Hong-wei4 *
    2015, 33(1):  7-35-39. 
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    Objective  To investigate the application value of empty puparia in species identification of common sarcosaphagous flies.  Methods  Fifty-five samples of adult flies and their empty puparia were collected. All the samples were identified as 2 families, 6 genera and 8 species by morphological characteristics. The samples were divided into 3 groups according to their time period between eclosion and our analyses: less than 2 years(n=23), 2-5 years(n=20), and more than 5 years(n=12). The mtDNA of each sample was extracted by CTAB method. The purity and concentration of DNA were tested. PCR products were amplified using two sets of primers. Two sequences of COⅠ gene(sequenceⅠ: 498 bp, sequenceⅡ: 841 bp) from each sample were compared to the sequences in GenBank using BLAST for species identification.  Results  The mtDNA was extracted successfully from all the samples. DNA concentration of adult chest muscle preserved less than or equal to 5 years and empty puparia preserved less than 2 years ranged from 1.0 to 3.0 μg/μl, and the value of A260/A280 ranged from 1.6 to 1.8. The purity and concentration was lower than 1.6 and 1.0 μg/μl, when the adult chest muscle and empty puparia preserved more than 5 years and 2 years, respectively. DNA concentration of the samples significantly decreased with the prolonged preservation time(P<0.01). Two sequences of COⅠ gene was amplified in adult chest muscle and empty puparia which preserved less than 2 years. The success rates of amplification decreased with the prolonged preservation time, especially for the sequence Ⅱ(P<0.01). The morphological identification  of 8 species did not match exactly with the results based on the COI gene, correct species identification occurred in 6 and 7 species out of 8 based on the two sequences, respectively, and their Max ident value exceeded 97%.  Conclusion  Empty puparium samples can be used to extract mtDNA and identify species.

    Effects of Omphalia lapidescens and Praziquantel on the Infectivity and Ultrastructure of Spirometra erinacei Plerocercoids
    SONG Guo-ping,LI Jin-fu,CHEN Yan*,XU Jing
    2015, 33(1):  8-40-44. 
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    Objective  To study the effects of Omphalia lapidescens and praziquantel on the infectivity and ultrastructure of Spironetra erinacei plerocercoids.  Methods  The plerocercoids were taken from frogs (Rana nigromaculata). A total of 168 mice were divided into 21 groups(8 mice per group), each of them was orally infected with 5 plerocercoids. The mice in group 1-9 were inoculated with plerocercoids cultured in media respectively containing different concentrations of O. lapidescens suspension (20, 40 or 80 mg/ml) for 4, 12 or 24 h, respectively. The mice in group 10-18 were inoculated with plerocercoids cultured in media respectively containing different concentrations of praziquantel(20, 80 or 320 μg/ml) for 4, 12 or 24 h, respectively. The mice in group 19-21 were inoculated with plerocercoids cultured in normal culture fluid for 4, 12 or 24 h, respectively, and served as controls. One week after infection, the mice were sacrificed to collect the plerocercoids. Worm reduction rate was calculated. The ultrastructure changes of plerocercoids were observed under transmission electron microscope(TEM) and scanning electron microscope(SEM), respectively.  Results  The average number of plerocercoids detected from mice infected by pleroceroids treated with 40, 80 mg/ml O. lapidescens suspension for 4, 12 or 24 h were 1.6, 1.0, and 0.3; 0.3, 0, and 0, respectively, and significantly lower than that of the infected controls(4.1, 3.5 and 3.3)(P<0.05); the worm reduction rates were 60.0%, 71.4%, and 90.1%; 92.7%, 100%, and 100%, respectively. The average number of pleroceroids detected from mice infected with pleroceroids treated with 320 μg/ml praziquantel for 4, 12, or 24 h were 1.9, 1.3, and 0.4, and significantly lower than that of the infected controls (P<0.05); the worm reduction rates were 53.7%, 62.9%, and 87.9%, and lower than that of 20 μg/ml praziquantel group(14.6%, 2.9%, and 6.1%) and 80 μg/ml praziquantel group(24.4%, 17.1%, and 24.2%)(P<0.05). The ultrastructure of plerocercoids cultured in 20 mg/ml O. lapidescens suspension, 20 or 80 μg/ml praziquantel for 4, 12 or 24 h had no significant difference compared with control groups. The plerocercoids cultured in 40 mg/ml O. lapidescens for 4 h or 320 μg/ml praziquantel for 4 or 12 h, showed mild contracture. The pleroceroids cultured in 40 mg/ml O. lapidescens for 12-24 h showed: agglutinate, fusion, fracture or abscission of microtriches, breakdown of plasma membrane, excretion of calcareous corpuscles, and tegument tissue damages. After cultured in 80 mg/ml of O. lapidescens for 24 h, the tissues of plerocercoid were damaged seriously. After cultured in 320 μg/ml praziquantel for 24 h, the plerocercoids showed: obvious contracture in the anterior end of plerocercoid, edema and bulge of plasma membrane, morphological changes of calcareous corpuscles, increase of secretory granules, glycogen depletion, and chromatin compaction in  flame cells.  Conclusion  The infectivity of Spironetra erinacei plerocercoids decreases along with the time of culture and the increase of drug concentration. Omphalia lapidescens and praziquantel can cause extensive tissue damage to the plerocercoids in vitro, and the effect of O. lapidescens on the infectivity and ultrastructure of plerocercoid is more considerable than that of praziquantel.

    Epidemic Status of Echinococcosis in Gannan Tibetan Autonomous Prefecture of Gansu Province during 2007-2011
    WANG Qing-hua,SHANG Wen-jie*,ZHAO Chun-tao,ZHANG Shu-wen,LU Shou-long,LIU Xiao-dong
    2015, 33(1):  9-45-48. 
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    Objective  To investigate the prevalence of echinococcosis in Gannan Tibetan Autonomous Prefecture of Gansu Province since the implementation of the echinococcosis control project from Sepical Funds for Central Government Subsidies to Local Public Health(2007-2011).  Methods  Eight counties of Hezuo, Lintan, Zhuoni, Luqu, Maqu, Xiahe, Zhouqu, and Diebu were selected as survey sites. The prevalence in the sampled population was investigated by B ultrasound examination. Hydatid infection in children below 12 years old was serologcially investigated by ELISA. The fecal samples from dogs were determined for Echinococcus infection by double antibody sandwich ELISA method. Livestock were dissected through slaughterhouse for pathological examination. Data of echinococcosis cases of Gannan Tibetan Autonomous Prefecture from 2007 to 2011 were collected from the National Infectious Diseases Reporting System, and statistically analyzed by using SPSS 10.0 and Epi info software.  Results  A total of 257 823 people received type B ultrasound examination in the 5 years. Five hundred eighty-one echinococcosis cases were found with an overall prevalence of 0.2%, including 578 cases of echinococcosis granulosus and 3 cases of echinococcosis multilocularis. The annual prevalence in the population decreased year by year, from 0.4% (97/21 938) in 2007 to 0.1% (68/63 980) in 2011 (P<0.05). Three hundred and six cases were officially reported to the National Infectious Diseases Reporting System during the period. Among those, female patients accounted for 58.2% (178/306) and male patients 41.8% (128/306). By occupation, more infection were found in herdsmen (82.0%, 251/306), followed by farmers(7.8%, 24/306). The cases were mainly distributed in the 20-60 year-old age group, with the highest prevalence in the group of 30-39 years(23.5%, 72/306). The sero-positive rate in children was 4.7% (1 571/33 613), which was highest in 2008 (8.4%, 413/4 907), and lowest in 2010 (3.2%, 223/7 021)(P<0.05). The mean positive rate of coproantigen in dogs was 6.3% (2 511/40 179), decreased from 11.9% (335/2 819) in 2007 to 6.3% (734/11 666) in 2011(P<0.05), with lowest positive rate in 2009 (3.7%, 354/9 550). The mean prevalence of livestock was 4.1% (914/22 087), decreased from 8.8% (235/2 658)in 2007 to 2.0%(144/7 347) in 2011(P<0.05).  Conclusion  Since the project implementation for echinococcosis control in 2007, the prevalence of hydatid desease in the population, the sero-positive rate in children, the positive rate of dog coproantigen, and the prevalence in livestock have been significantly decreased.

    pplication of Nested PCR in the Diagnosis of Imported Plasmodium Ovale Infection
    HUANG Bing-cheng,XU Chao,LI Jin,XIAO Ting,YIN Kun,LIU Gong-zhen,WANG Wei-yan,ZHAO Gui-hua,WEI Yan-bin,WANG Yong-bin,ZHAO Chang-lei,WEI Qing-kuan*
    2015, 33(1):  10-49-51. 
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    Objective  To identity Plasmodium ovale infection by 18S rRNA gene nested PCR.  Methods  Whole blood and filter paper blood samples of malaria patients in Shandong Province were collected during 2012-2013. The parasites were observed under a microscope with Giemsa staining. The genome DNA of blood samples were extracted as PCR templates. Genus- and species-specific primers were designed according to the Plasmodium 18S rRNA gene sequences. Plasmodium ovale-positive specimens were identified by nested PCR as well as verified by sequencing.  Results  There were 7 imported cases of P. ovale infection in the province during 2012-2013. Nested PCR results showed that the P. ovale specific band (800 bp) was amplified in all the 7 specimens. Blast results indicated that the PCR products were consistent with the Plasmodium ovale reference sequence in GenBank.  Conclusion  Seven imported cases of ovale malaria in Shandong Province in 2012-2013 are confirmed by nested PCR.

    Research Progress on the Molecular Mechanisms of Mosquito Innate Immunity
    GUO Xiu-xia,WANG Huai-wei *
    2015, 33(1):  11-52-57. 
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    As the important vector of the mosquito-borne diseases, mosquitos rely on their innate immune system against pathogens infection, which includes cellular immunity and humoral immunity. Both of them are interacted and coordinated, via the pattern recognition receptors, immune signaling, antimicrobial peptides, phenoloxidase-based melanization, phagocytosis, and so on. This paper reviews the research progress on the molecular mechanisms of mosquito innate immunity.

    Controlled Veterinary Drug Delivery Systems against Parasitic Infection
    TU Zhen,ZHANG Hao-bing*
    2015, 33(1):  12-58-63. 
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    Parasitic infections, especially the gastrointestinal and lung nematode infections, are most common in livestock in temperate areas, and it is the major constraints affecting livestock production. In grazing season, outdoor activities of animals cause inconvenience to the application of antiparasitic drugs. Therefore, controlled drug delivery systems can prolong the effect time and reduce the difficulty of drug administration. This review summarizes several types of long-term delivery devices and dosage forms including intraruminal devices, long-acting injectables, in-situ forming implants, novel microparticles and nanoparticles. Their advantages and drawbacks are dicussed.

    Progresses on Antitumor Immune Mechanisms of Protozoon
    HUANG Hai-bin,YANG Wen-tao,WANG Chun-feng,YANG Gui-lian*
    2015, 33(1):  13-64-67. 
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    A variety of protozoons(Amoeba, Trypanosoma cruzi, Eimeria, Plasmodium falciparum and Toxoplasma gondii, etc.) can activate and regulate the human immune system, change tumor-induced immunosuppression, and enhance the anti-tumor immune response. This paper reviews the effects of protozoon on cellular and humoral immunity, the tumor microenvironment in tumor-bearing organisms, and reveals the antitumor immune mechanisms of protozoon.

    Research Progress and Application Future of MicroRNA-36
    FENG Sheng-yong,SHAO Chang-chun,ZHU Xing-quan,XU Min-jun*
    2015, 33(1):  14-68-71. 
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    MicroRNA-36(miR-36) is a recently discovered miRNA family which including at least eight members, and specifically existed in helminths compared with other miRNAs that widely exists in almost all kinds of lives. This paper reviews recent research advances about miR-36 to provide further fundamental information for helminth and miRNA study.

    Application of CT and MRI in the Diagnosis of Paragonimiasis in Brain and Spinal Cord
    LI Qiang1,2,QIN Da-ming3 *
    2015, 33(1):  15-18-20. 
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    Clinical data of 22 cases with paragonimiasis in brain and spinal cord in the Central Hospital of Enshi Tujia and Miao Autonomous Prefecture during 2003-2012 were retrospectively analyzed. Twenty-two cases aged from 6 to 17 years old, including 15 males and 7 females, were from the countryside, and had a history of eating raw crabs. CT and MRI showed that all the cases had cerebral lesion, 16 cases had unilateral lesion of cerebral hemisphere, and 6 cases had bilateral hemisphere lesions. There were mainly two kinds of imaging changes: ① the infarct and low-density edema of large area with focal hemorrhage; ② annular lesions. Among the 6 cases with intracerebral hematoma, 2 patients received surgical removal, and all the patients were treated with praziquantel, 25 mg/kg each time, 3 times per day, 3 d for a course. This regimen was repeated at a 7-day interval. All the patients were cured and discharged from hospital.

    Observation of the Feline Intestinal Epithelial Cell Infected with Toxoplasma gondii
    ZENG Jun-ling1,NA Ren-hua2,CHEN Ai-yuan2,WEI Hai-xia2,YAO Yun-ying2,PENG Hong-juan2,YANG Pei-liang 1*,CHEN Xiao-guang2
    2015, 33(1):  16-32-34. 
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    Small intestine samples of neonatal cat were aseptically collected from the jejunum-ileum region and digested with collagenaseⅪ/dispaseⅠ. Immunohistochemistry results showed that feline intestinal epithelial cells were successfully isolated and could be cultured. Cytokeratin was positive in the cytoplasm of feline intestinal epithelial cells. The cells were infected with the bradyzoites of Toxoplasma gondii Prugniaud strain, and the rupture of the cells was observed on the 72nd day post-infection. The sexual stage of T. gondii did not occur, however.

    Diagnosis of an Imported Case of Plasmodium ovale Infection in Nanping City,Fujian
    ZHANG Zhi-ping1,ZHANG Cong-hui2,ZHUO Ming-ying1,LIN Yao-ying3,WEI Hui-zheng4,ZHU Huai-min2 *
    2015, 33(1):  17-72-74. 
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    An imported case previously misdiagnosed as vivax malaria was reviewed. The epidemiological data and blood sample were collected. The detection was conducted by microscopy, rapid diagnostic test(RDT) and nested PCR. The case was finally comfirmed as the first imported case of Plasmodium ovale infection in Nanping.

    mRNA Expression Analysis of 10 Reproduction-related Genes in Different Developmental Proglottids of Moniezia expansa
    WANG Zheng-rong1,BO Xin-wen1 *,ZHANG Yan-yan1,MA Xun2
    2015, 33(1):  18-75-77. 
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    According to the results of transcriptome sequencing in Moniezia expansa, 10 functional genes which represent different expression patterns in different developmental proglottids were selected, including KIFC1, Kif17, tgf-beta, SmadD, tgf-beta receptor, HSD5, aminopeptidase puromycin, Methionine aminopeptidase 2, transcription factor fork head and Sox transcription factor. A real-time fluorescent quantitative PCR assay was developed for detection of mRNA expression level of these taget genes with its beta-tubulin as an internal control. The results showed that compared to scolex-neck proglottids, the mRNA levels of 2 genes(KIFC1, Kif17) in immature proglottides, 4 genes(KIFC1, Kif17, tgf-beta receptor, and aminopeptidase puromycin) in mature proglottides, and 3 genes(HSD5, tgf-beta receptor, and Methionine aminopeptidase 2) in gravid proglottides were up-regulated (P<0.01).