Table of Content

31 October 1998, Volume 16 Issue 5

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论著

THE ROLE OF L ARGININE AND L CITRULLINE IN ACTIVATED MACROPHAGE AGAINST TOXOPLASMA GONDII INFECTION IN VITRO

ZhengChunfu;LinJianyin

1998, 16(5):  326-330. 

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AIM: To determine whether both the production of NO and the toxoplasmastatic or toxoplasmacidal activity of activated macrophages cultured in physiologic levels of arginine can be enhanced by increasing the availability of arginine, or citrulline. METHODS: After the activated Mφ were infected with the RH strain of T.gondii ,the levels of NO production, the infection rate of Mφ and the number of tachyzoites in parasitophorous vacuoles were determined after the Mφ were cultured in medium added with various concentrations of L-arginine or L-citrulline for 18 h. RESULTS: (1) The multiplication of intracelluar toxoplasmic tachyzoites could be inhibited by activated macrophages, depending on the production of NO in the presence of physiologic levels of arginine. (2) Increased exogenous arginine or citrulline resulted in a significant elevation of NO producion induced by activated macrophages and further reduction of infection rate of macrophages as well as inhibition of multiplication of intracelluar tachyzoites. (3) Citrulline could fully substitute for arginine in enhancing NO production and toxoplasmastatic or toxopasmacidal activity. CONCLUSION: The physiological levels of arginine were able to induce sufficient intrinsic NO production to inhibit intracellular
multiplication of toxoplasmatic tachyzoite but unable to protect cell from infection and that
increasing the substrate levels for NO biosynthesis may enhance in vitro toxoplasmastatic or
toxoplasmacidal activity of activated macrophages.

GENOTYPING OF PLASMODIUM FALCIPARUM ISOLATES BY AMPLIFICATION OF GLUTAMATE RICH PROTEIN GENE USING POLYMERASE CHAIN REACTION

ZhuXinping;GeorgesSnounou;WilliamJarra;SodsriThaithong;K.NeilBrown

1998, 16(5):  331-334. 

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AIM: To develop a genotyping method based on amplifying glutamate rich protein
(GLURP) gene for the diagnosis and identification of Plasmodium falciparum . METHODS:Two pairs
of primers specific for GLURP gene of P.falciparum were designed and synthesized.R 2 polymorphic
domain of GLURP gene was amplified by nested PCR, which was applied to genotyping of
P.falciparum isolates obtained from patients attending the malaria clinic at the village of
Borai, Thailand. RESULTS: Conspicuous polymorphisism of GLURP alleles in natural populations of P. falciparum was found. 290 GLURP alleles were detected in 154 P. falciparum infections. Among the above-mentioned alleles, 12 different GLURP genotypes were distinguished according to different DNA sizes. Of them , the most frequently found allele was a variant of 770 bp , the least allele was that of 1 100 bp. More than 43% of the patients were found to be infected with mixed alleles. No apparent change for frequencies of the 12 different alleles was found in the 9-month longitudinal study. CONCLUSION:A genotyping method is developed for the research of strain taxonomy and pathogenesis of malaria parasites.

IN VITRO AND IN VIVO EFFECT OF LEVOPRAZIQUANTEL, DEXTROPRAZIQUANTEL VERSUS RACEMIC PRAZIQUANTEL ON DIFFERENT DEVELOPMENTAL STAGES OF SCHISTOSOMA JAPONICUM

XiaoShuhua;YouJiqing;MeiJinyan;HuYuqin;ZhouDehan;BrianA.Catto

1998, 16(5):  335-341. 

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AIM: To compare the antischistosomal effect of racemic praziquantel (Pra) and its
enantiomers, levopraziquantel (L Pra) and dextropraziquantel (D Pra), on different developmental
stages of Schistosoma japonicum. METHODS: The in vitro effects of the drugs were determined in
different stages of schistosomes maintained in RPMI 1640 supplemented with 20% calf serum. In
vivo study mice infected with schistosome cercariae were treated intragastrically (ig) with Pra,
L-Pra or D-Pra at different intervals after infection. The efficacy of the drugs was evaluated by residualmean worm number. RESULTS: Based on the degree of tegument damage induced by L-Pra, d28 and d35 schistosomes were most susceptible to L-Pra, while d14 schistosomules being least susceptible. At comparable concentrations of 0.1- 1 g/ml, L-Pra was more active than Pra even when the concentration of L-Pra was reduced to one-half of the minimum effective concentration Of Pra. At above-mentioned concentrations D-Pra exhibited no apparent in vitro effect on different stages of schistosomes. When infected mice were treated ig with L-Pra, Pra or D-Pra at a single dose of 300mg/kg or 500 mg/kg, only the former two drugs showed apparent effect on d0, d21, d28 and d35 schistosomes and less or much less effect on d3, d7 and d14 schistosomules. D-Pra only exhibited a negligible effect on d35 adult schistosomes as compared with L-Pra and Pra. When mice infected with d35 adult schistosmes were treated ig with L-Pra 150 mg/kg, the efficacy was similar to that of mice treated with Pra 300 mg/kg. CONCLUSION: L-Pra is the principal active component against schistosomes in racemic Pra.

CLONING AND SEQUENCE ANALYSIS OF THE GENE ENCODING THE PARTIAL REGION CS PROTEIN OF A PLASMODIUM FALCIPARUM ISOLATE FROM YUNNAN

XiaoJianhua*;LiMing;ChuiDong;BiHuixiang;WangPing;LiYingjie

1998, 16(5):  342-346. 

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AIM:Determining nucleotide sequence of the circumsporozoite protein partial gene of
the Plasmodium falciparum PFD-3/YN(Yunnan of China)and finding out the differences of the CS
gene sequence between Chinese Plasmodium falciparum isolate and other isolates.METHODS:The
circumsporozoite protein gene fragment was amplified by polymerase chain reaction and cloned
into M13 bacteriophage.M13 CSP single strand DNAs of the three positive clones were extracted
respectively.Then,the nucleotide sequence of the CS gene ragment was determined by the dideoxy chain termination method. PCGENE software was used to compare and analyze the CS gene sequence of the six isolates. RESULTS:Different degrees of diversity of the CS gene sequences were found among P. falciparum PFD - 3/YN and other isolates (T4,Wellcome,NF54, 3D7 and 7G8). A non-silent substitution at the nucleotide level being found in the P. f Th/Tc antigenic epitope region. CONCLUSION: There were differences in the CS gene sequence among P. falciparum PFD- 3/YN and those of other isolates.

STUDIES ON THE LIFE CYCLE OF PARAGONIMUS HETEROTREMUS

HuWenqing

1998, 16(5):  347-352. 

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AIM:In order to clarify the biological features of the lung fluke, Paragonimus
heterotremus , it is necessary to replicate its complete life cycle in the laboratory. METHODS:
Tricula wumingensis infected with the miracidiae of P.heterotremus were regularly dissected
after infection for larvae observation. The cercariae of P. heterotremus from positive snails
were used for experimental infection of the crab, Samanniathelphusa nanningensis . The crabs
were then examined for the metacercariae at regular in tervals. The infection of mammals (cats and rats) was performed with the metacercariae obtained from exerimentally infected crabs. RESULTS: The miracidia were hatched by incubating the eggs in water (room temperature 20 ℃ to 31 ℃) for 16 days to 21 days. The epidermal cells of the miracidia were arranged in 4 transverse rows, 6 in the firstrow , 7 in the second, 3 in the third, and a single terminal cell at the posterior end of the body. The cells of the first row have indentation in their bases. Mature sporocysts, first generation rediae and second generation rediae (containing dissociative cercariae) ,were found on the 26th day, 41st day and 58th day after infection of the snails respectively ( room temperature 21℃- 30℃). The sporocyst had a birth pore at its anterior tip. The length ratio of the gut to the body was 1∶3 in the first and 1∶6 in the second generation rediae. The mature second generation rediae contains 5- 21 mature cercariae which emerge from its pharynx. The cercaria has general characteristics of Paragonimus. Its flame cell pattern was formulated as 2[ (2+ 2+ 2) + (2+ 2+ 2) ]= 24. After the infection of crabs with cercariae, thematu remetacercariae were found in the crabs on the 60th day. The crabs could be successfully infected by either: giving mature cercariae orally to them or raising them in water with postive snails. After the metacercariae obtained from experimental crabs were given to cats and rats, P. heterotremus adult worms were recovered from the worm - cyst in their lungs. The morphology of various larval stages and adult worms were described. CONCLUSION: The life cycle of P.heterotremus was completed for the first time in the laboratory. The whole life cycle from egg to adult worm has been described in the present study.

ESTABLISHMENT OF GERMINAL CELL LINE OF ECHINOCOCCUS GRANULOSUS

LiuJinming;GuHuiming;WangAihua;ChenYanjun

1998, 16(5):  353-356. 

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AIM:To establish a cell line of Echinococcus granulosus. METHODS:The proliferating
membranes striped from liver cysts in a naturally infected sheep from Urumq were released to
monodispersed germinal cells by grinding. The germinal cells were cultivated in the RPMI 1640
medium supplemented with 10%—20% calf serum on collagen coated 24 well culture plate
alternately from passage 1 to passage 14,and then continued. The morphological feature and
growth situation were observed by light microscopy. The cultured cells were inoculated into BALB/c mice to identify the infectivity . ELISA was used to determine the immunogenicity of the cells . RESULTS: The germinal cells have been cultivated continuously up to passage 75. The subcultured cells were circular in shape with smooth surface and had the tendency to form syncytia and tissue-like masses. The cells from this cell line could be stored for at least 15 days in refrigerator at 4℃ and 10 months in liquid nitrogen. No cystmaterials were detected in the mice inoculated with cells. The antigens from cell line could react with positive sera from mice infected with protoscoleces and sera against secreted antigens of cystmembrane , soLuble antigens from cystmembrane, soluble antigens from protoscoleces and SHF. CONCLUSION: A germinal cell line of Echinococcus granulosus was successfully established.

STUDIES ON DYNAMIC CHANGES OF SOD AND MDA IN CULTURED CELLS FROM SCHISTOSOMA JAPONICUM SCHISTOSOMULA

JiangMingsen;ChenXiaobei*;DongHuifen;YangMingyi;NiYonghui;ZhouShulong

1998, 16(5):  357-359. 

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AIM:To observe the dynamic changes of superoxide dismutase(SOD) and

maldondialdehyde(MDA) in cultured cells from Schistosoma japonicum schistosomula and to explore

the degeneration process of cultured cells. METHODS: Pyrogallol method and thiobarbituric acid

method were used to determinate the levels of SOD and MDA , respectively,in the cells cultured

for 0-54 days. RESULTS: SOD in the cells reached a peak level on d12 of the early stage of

culture and then decrease. The MDA levels in the cultures cells ascended gradually, and had two peaks on d18 and d48, during the period of culture. CONCLUSION: The cultured cells from S. japonicum schistosomula degenerated progressively.

IMMUNODIAGNOSIS OF CYSTICERCOSIS BY SIMPLIFIED WESTERN BLOTTING USING RECOMBINANT FUSION PROTEIN AS ANTIGEN

PengYucong;ChenRuiwen;SunShuhan;DaiJianxin;WangJunxia

1998, 16(5):  360-363. 

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AIM:To provide simple and useful method for the immunodiagosis of cysticercosis.METHODS:Fusion protein of β galactosidase Cysticercus cellulosae cDNA was
analysed with simplified Western blot.RESULTS:The positive rate was 93.5% when detecting 124
cases of human cysticercosis sera with four clones (cC1,cC2,cP1 and cH1) and 100% when detecting
38 cases of porcine cysticercosis sera with three clones (cC1,cC2 and cP1),being higher than those using monoclone fusion protein alone. Moreover, the fusion proteins did not react with other parastitosis sera. CONCLUSION: Simplified Western blot analysis using recombinant fusion protein as antigen was highly sensitive and specific and easy to be operated.

ULTRASTRUCTURAL OBSERVATIONS ON THE FORMATION AND METABOLISM OF CALCAREOUS CORPUSCLES IN CYSTICERCUS CELLULOSAE

TianXifeng;ZhangBaodong

1998, 16(5):  364-367. 

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AIM : To study the formation and metabolism of calcareous corpuscles from Cysticercus cellulosae at the ultrastructure level. METHODS: Transmission electron microscopy. RESULTS: The developmental processes of calcareous corpuscles could be divided into two stages: the intracellular formation stage and the extracellular metabolic stage. The calcareous corpuscles were formed in a cell which we named calcareous corpuscle forming cell. At the early stage of the formation, the corpuscles appeared to be secretory granules in the cells. With the development of the co rpuscles, they became bead-shaped and lamellae-like, then the calcareous corpuscle forming cell enlarged and the organellae degenerated. Finally the corpuscles gathered to form particle substances with black dense background,while the nucleus and organellae of the forming cell all disappeared . There were 1- 3 or 10- 20 calcareous corpuscles in a mature forming cell. Then, the corpuscles were released to the parenchymal tissues and gradually appeared to be concentric lamella or an empty cavity during the metabolic process . CONCLUSION : The calcareous corpuscles were formed in calcareous corpuscle forming cell and consumed in metabolic process in the parenchymal layer of Cysticercus cellulosae.

OBSERVATION ON THE INFECTIVITY OF DIFFERENT DENSITIES OF PLASMODIUM VIVAX TO ANOPHELES SINENSIS

LiHuaxian;YangBailin;WangWenren;HuHuixian;WangWenjun;WangXiang;LiXinglian;HuangGuozhen;liChongzhen

1998, 16(5):  368-371. 

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AIM: To study the infectivity of different densities of P.vivax to An.sinensis. METHODS:Venous blood samples from 38 volunteers artificially infected with P.vivax of South Yunnan collected at the time of initial parasitaemia, primary attack and relapse, were used to infect Anopheles sinensis by feeding on parasitaemia blood in vitro. The finding of oocysts in the mid gut of the mosquito by microscope was taken as criteria of positive infection. RESULTS: P. vivax in the initial parasitaemia did not infect the mosquito . In the cases of primary attack, when the density of parasite was more than 100/μl blood, the infectivity and intensity rose with the prolongation of the course of the disease and the increase in the density of the parasite . The infect ivity of the P. v ivax in the relap se cases w as h igher than in the
primary attack cases, the mosquitoes could be infected at a parasite density of 1/μl blood.
The mosquitoes acquired a higher infection rate, a higher positive rate of mosquito mid-gut and a higher oocyst index in the primary attack cases whose parasite density were higher than 1 000/μl blood and in the relapse cases whose parasite density were higher than 100/μl blood. CONCLUSION: The P. vivax in the period of clinical attack was one of the most dangerous period of the spread of malaria . The cases of relapse were the more dangerous infective sources in malaria transmission.

SCREENING AND CHARACTERIZATION OF SCHISTOSOMA JAPONICUM REINFECTION RELATED cDNA CLONES

WuHaiwei;WangRongzhi;ChenShuzhen;SuChuan;ZhangZhaosong;WuGuanling

1998, 16(5):  372-375. 

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AIM:To obtain cDNA clones coding for S. japonicum(Sj) reinfection related specific
immunologic molecules for vaccination or diagnosis. METHODS: An adult S.japonicum cDNA library,
of which the cloning efficiency is 3.13×10 6, with directional inserts in the vector lambda
gt11, was screened with pooled serum from 43 reinfected human cases in a schistosomiasis endemic
area with high level of Sj specific lgG4 antibodies. Primers located several bases away from the insert position on the lambda gt11 arm were used to amplify the insert fragments by standard PCR. SILVER SEQUENCETM (PCR-silver) DNA sequencing system was then used to sequence selected positive clones with relatively large inserts and homology comparison with GenBank database was also carried out. RESULTS AND CONCLUSION: Eleven phage plaques were defined as positive out of the 8. 6×104 phage plaques immunoscreened by human specific lgG4 antibody. GenBank database retrieval showed that 4 new Sj gene clones including Sj reinfection-related specific protein-encoding gene and Sj mitochondrial protein-coding gene have been obtained.

实验研究

CONTINUOUS CULTIVATION OF A LARGE NUMBER OF PLASMODIUM FALCIPARUM GAMETOCYTES IN CARBON DIOXIDE INCUBATOR *

XuXuenian;FengZheng;YangBailin;WangJujun;HuWei;NiQizhen

1998, 16(5):  376-379. 

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AIM: To establish a method for continuously cultivating a large number of P.
falciparum gametocytes in vitro using carbon dioxide incubator.METHODS: The number of
gametocytes produced in experimental and control groups was compared after the addition of
various concentrations of NaHCO 3 to the culture medium. RESULTS:The gametocytes began to rise
on day 5 of cultivation and reached a peak on day 11-13. The peak gametocyte loads were 1.9%±
0.6% and 1.3%±0.4%( P <0.05) in experimental and control group , respectively, indicating that the complete medium with 30 mmol/L sodium bicarbonate was benificial to gametogenesis. The numbers of stages I - V gametocytes rose to a peak on d5, d7, d11, d13 and d15, respectively. On day 15 the percentage of stage V gametocytesw as 7. 1% - 52. 6% w ith an average of 24. 3%. The ratio of macrogametocyte to microgametocyte was 12. 8∶1. The parasites were able to produce high gametocytaemia up to 24th subculture after thawing. Laboratory reared Anopheles stephensi fed through membrane on blood infected with P. falciparum were disected, no oocyst was found in the midgut. CONCLUSION: A culture system which could consistently and stably produce a large number of gametocytes of P. falciparum was established.

DETECTION OF TOXOPLASMA GONDII DNA IN HUMAN LYMPH NODE TISSUE BY IN SITU HYBRIDIZATION

LiuCuimei;OuyangKe;TanDeming;ZhangZheng

1998, 16(5):  380-383. 

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AIM: To detect the presence of Toxoplasma gondii in lymph node tissue in patients
with Toxoplasma infection. METHODS: T.gondii ( RH strain ) specific DNA fragment clones were
obtained by using PCR and gene recombination technique. The DNA fragments used as hybridization
probes were labelled with digoxigenin by random primer method. The technique of in situ
hybridization (ISH) was used to detect T.g DNA in the lymph node sections. RESULTS: Four out of 120 samples T. g DNA were found positive, one with Hodgkins disease (HD ) (1/32) , one with non-Hodgkin’s lymphoma (NHL ) (1/41) and 2 with chronic lymphadenitis (CL ) (2/47). The total positive rate was 3.3%. It was demonstrated that this highly specific probe could detect 10 pg of the total RH strain T. g DNA. CONCLUSION: ISH was applicable in detecting pathogens in the lymphnode tissues of individuals with Toxoplasma infection.

防治经验

AN EPIDEMIOLOGICAL SURVEY OF SCHISTOSOME CERCARIAL DERMATITIS AMONG THE RESIDENTS LIVING ALONG THE BANKS OF THE HUAIHE RIVER SYSTEM

LiChaopin;MaChangling;GuJianzhong;LuWangying

1998, 16(5):  384-387. 

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AIM: To make an epidemiological survey of schistosome cercarial dermatitis among
the residents living along the banks of the Huaihe River. METHODS: Making inquiries about signs
and symptoms of schistosome cercarial dermatitis, and physical examinations of manifestation in
the residents (fishermen and peasants). Cercariae shed from the Radix auricularia collected in
the field were used to infect ducklings. The eggs and miracidia separated from the feces of the
full grown ducks bred by fishermen were used to infect Radix auricularia. RESULTS: Schistosome dermatitis locally known as“duck itch”(meaning duck-feces-dermatitis) was discovered among the residents living along the Huaihe River bank. The main signs and symptoms of the dermatitis included diffuse erythema or red papular eruption with areorae、urticae、urticant itch or titillation around the affected areas ( including thoracic part, abdomen and lower limbs etc). The cercariae with eye spot were found from Radix auricularia collected from Huaihe riverside and were identified to be the species of Trichobilharzia can both hatch miracidium , which laboratory-reared Radix auricularia were infected with miracidia hatched from the two types of eggs, rhomboid eggs and crescent eggs, found in the feces of full grown ducks bred by fishermen and shed the same cereariae with eye spot. Besides, adult worms of Trichobiharzia were obtained by disecting both experimentally infected duckilings bred in the laboratory and naturally infected ducks bred in the Huaihe riverside area. The above-mentioned eggs, miracidia, cercariae and adult worms were all identified to belong to the life-phase of Trichobilharzia. CONCLUSION: The epidemic disease known as schistosoma cercarial dermatitis harmful to the health of the residents living along the banks of Huaihe River is caused by the cercariae of Trichobilharzia.