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Table of Content

    31 May 1994, Volume 12 Issue 2
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    IN VITRO CULTIVATION OF THE EXOERYTHROCYTIC STAGE OF PLASMODIUM VIVAX (SOUTHERN CHINA ISOLATE)
    1994, 12(2):  81-84. 
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    An in vitro culture system for the exoerythrocytic(EE)stage of Plasmodium vivax was first developed in our laboratory in China. Anopheles stephensi mosquitoes were infected by membrane feeding with heparinized blood from a volunteer.After 14 18 days,the mosquito salivary glands were aseptically dissected in culture medium and ground in a tissue grinder to form sporozoite suspension.Sporozoites were counted and added to the cultures of monolayer hepatoma cells at the number of 4.9 ×10 4-1 3×10 5 per cover glass.Sometimes,8 10 pairs of infected gland were added directly to the cultured cells.On day 7,EE schizonts of P.vivax were found in cultures.In addition,normal erythrocytes (type o)were added to the cultures on day 10 at a concentration of 10 8 per dish.Fourteen days later,erythrocytes in culture supernatant were collected and thin blood films were made.Numerous intra erythrocytic P.vivax parasites were identified on the films after Giemsa staining. Most intra erythrocytic forms were rings and early large trophozoites.Schuffers dots were present in many of the infected cells which were pale and obviously enlarged.These results indicated that the in vitro hepatic cycle of P.vivax was establisthed.
    DNA SEQUENCING OF CIRCUMSPOROZOITE PROTEIN GENES OF PLASMODIUM VIVAX FROM FOUR DIFFERENT COUNTRIES IN WEST PACIFIC REGION: COMPARATIVE STUDY ON THE FLANK SEQUENCES
    1994, 12(2):  85-91. 
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    P.vivax CSP gene of 18 isolates from infected blood of patients living in China,Philippines,Solomon Islands and Papua New Guinea in West Pacific region has been sequenced from both terminal end.The total readable sequence in most isolates was about 725 base pair (bp),including the first two repeat units (70-330 bp) to the N terminus,and the last two repeat units to the C terminus (763-1 228 bp). Comparison and analysis of these obtained sequences with the published sequences of N.K., Thai., Belem,Sal l.and BZL strains showed that:the N and C terminus sequences flanking the centre repeats in CSP gene were highly conserved in all isolates and identical with the 5 published sequences but a double base pair substitution in each end and a remar kable polymorphism in the postrepeat variable region in C terminus were found,including some diversities with obvious geographic characteristics which have not been reported pre viously .
    ANALYSIS OF THE TARGET EPITOPES RECOGNIZED BY TWO MONOCLONAL ANTIBODIES DIRECTED TO EGG ASSOCIATED FRACTIONS OF SCHISTOSOMA JAPONICUM
    1994, 12(2):  93-99. 
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    Two IgM isotype monoclonal antibodies(McAb),2H10 and 2H1,recognizing repetitive epitopes on Schistosoma egg associated molecules were characterized and their specificities were identified in a two site sandwich ELISA system.In consistent with the differences in immunological behaviour and specificity demonstrated with immunoelectrophoresis(IEP) and immunofluorescent antibody(IFA) techniques,absolutely negative reactions found in the he terologous detecting system with alternated capture and detecting McAbs of the two revealed a complete incompatibility giving evidences that epitopes on different molecules were recognized.Immunological liability of the target antigen SEA or SEA TCA to the two McAbs were demonstrated on sodium periodate and trifluoroacetic acid treatment indicating the biochemical nature of these epitopes were glycosyolated molecules with apparently higher resistance to the oxidizing agent showing in 2H10 recognizing epitopes. By means of an ion gradient Mono Q FPLC system (Pharmacia),2H10 reactive epitopes of SEA,being tested not so efficiently adsorbed by ConA sepharose affinity column,was found successfully concentrated in the profile eluted with pH 8.0 PBS at 0.2 0.4 NaCl ionic strength. Repeated trials on SDS PAGE and Western blotting analysis with the reactive fractions further showed a heterogeneity of molecular weight range as well as the non transferable property of the CHO reactive groups.
    MORPHOLOGY AND PHAGOCYTIC ACTIVITY OF HEMOCYTES OF ONCOMELANIA HUPENSIS
    1994, 12(2):  100-103. 
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    Hemocytes from Oncomelania hupensis hemolymph were studied in vitro. Spreading amoebocytes were the most common cell type.The average sizes of amoebocytes from Anhui and Yunnan were 8.42±0.73×7.46±0.52 μm and 7.63±0.65×6.85±0.54 μm, respectively. The nucleus was round in shape,and eosinophile granular inclusions were found in the endocytoplasm.Contacting with slide,the cells became flattened with extending pseudopodia. The ability of amoebocytes from snails of both localities to phagocytose sheep red blood cells was confirmed in vitro. The method to collect hemolymph from Oncomelania hupensis was developed, and the promise of this method in studying host parasite relationship as well as epidemiological investigation of schistosomiasis japonica in the mainland of China was discussed.
    PRELIMINARY STUDY ON EXPRESSION OF LEISHMANIA DONOVANI ANTIGEN GENE IN E.COLI
    1994, 12(2):  107-110. 
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    We constructed previously the expressing library of Leishmania donovani genomic DNA with λgt11 as vector.In this paper, 2×10 4 phages were plated on E. coli Y 1090r , and screened with a rabbit antiserum prepared by immunization with Leishmania donovani promastigotes.Bound antibodies were detected using alkaline phosphatase labeled anti rabbit antibodies.A positive expressing clone was detected and transferred into E.coli Y 1098r to prepare lysate,a 39 kDa polypeptide in E.coli Y 1089r lysate was recognized by anti Leishmania donovani serum.The result indicated that the 39 kDa polypeptide which was not fused with the major portion of β galactosidase existed disconnectedly.This finding remained to be further studied.
    DETECTION OF PLASMODIUM FALCIPARUM BY POLYMERASE CHAIN REACTION (PCR)
    1994, 12(2):  111-114. 
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    A pair of primers were designed and synthesized based on the DNA fragment coded erythrocyte binding antigen (EBA 175) of Plasmodium falciparum ( P.f .) and polymerase chain reaction(PCR) was performed to detect P.f. cultured in vitro .The amplified products were analysed by agarose gel electrophoresis and a specific 492 bp DNA band was showed in P.f. infected blood sample,but no band showed in P.vivax, P.cynomolgi , P.yoelii and P.berghei infected blood samples,nor in normal one.The parasite densities detected by this method could be as minimal as 10 parasites/20μl blood.The PCR technique showed high sensitivity and specificity.
    LABORATORY AND CLINICAL STUDY ON PNEUMOCYSTIS CARINII Ⅱ.IMMUNOBLOT ANALYSIS FOR DETECTION OF ANTI PNEUMOCYSTIS CARINII ANTIBODIES IN THE SERA FROM HEALTHY BLOOD DONORS
    1994, 12(2):  115-118. 
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    Rat Pneumocystis carinii cyst soluble antigen (CSA) was analysed by SDS-PAGE,and more than 20 polypeptide bands were found.The molecular weights of the major bands were 100kDa,85-94kDa,67kDa,52kDa and 34kDa,respectively.The specific IgG antibodies in the sera from healthy blood donors recognizing the 110kDa,105kDa,85kDa,67kDa,52kDa and 46kDa polypeptide antigens were identified by immunoblot analysis.The total positive rate of IgG specific antibodies was 56.7%(59/104).The 85kDa polypeptide antigen could also react with the monoclonal antibody derived from human Pneumocystis carinii .The positive rate of the specific anti 85kDa IgG antibody was 37.5%(39/104),but the specific anti Pneumocystis carinii IgM antibodies were not detected in all the sera examined.Three of 7 serum samples from the patients with Pneumocystis carinii pneumonia were positive for specific anti 85kDa IgG antibody(Figs 1 4).
    DETECTION OF SPECIFIC DNA FRAGMENT OF PLASMODIUM FALCIPARUM BY SOLID PHASE PCR
    1994, 12(2):  126-128. 
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    Solid phase PCR for detecting Plasmodium falciparum was established and used to detect cultured FCCl/HN isolate and malaria patients in Yunnan Province.The results revealed that the sensitivity of the method for detecting FCCl/HN isolate DNA was as minimal as 0.2 pg,or about 10 parasites. The specificity of the method was confirmed by discriminating malaria patients infected with different species of Plasmodium .The results suggest that solid phase PCR is a specific and sensitive method for early detection of P.falciparum.
    ULTRASTRUCTURAL CHANGE OF BRAIN GANGLION OF ONCOMELANIA HUPENSIS AFTER IMMERSION IN SODIUM PENTACHLOROPHENATE
    1994, 12(2):  129-132. 
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    The ultrastructure of the fibrous sheath,the layer of ganglionic cell bodies and the neuropil of brain ganglion of Oncomelania hupensis was first observed under transmission electron microscope. Ultrastructural changes were found in the brain ganglion after immersing the snails in sodium pentachlorophenate.The mitochondria of nerve fibers and neurons showed denaturation and necrosis,and glycogen in neurones was markedly reduced;the neuropil also exhibited denaturation and necrosis.The ultrastructural findings may explicate in some way the mechanism of molluscicidal action of NaPcP on Oncomelania hupensis via its action on the nervous system(Figs.1-6).
    EFFECTS OF MONOCLONAL ANTIBODIES ON PHAGOCYTOSIS OF ENTAMOEBA HISTOLYTICA TO HUMAN ERYTHROCYTES
    1994, 12(2):  138-141. 
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    Two monoclonal antibodies 3F7 and 4G6 were produced respectively against plasma membrane and nucleus cytoplasm of Entamoeba histolytica .The erythrocytophagous quantification and ability of pathogenic strain HM 1∶IMSS were evaluated by microscopy immediately at 5 min and 10 min after incubation with McAb 3F7 and McAb 4G6. Sera from normal mice and monoclonal antibody TCE 04 against Trypanosoma cruzi were prepared as negative controls.The McAb 3F7 showed stronger inhibitory action on the erythrocytophagous effect of HM 1∶IMSS than the McAb 4G6.The result suggested a significant inhibition( P 0.001) on erythrocytophagous effect of amoebic trophozoites in the presence of McAb 3F7. This inhibitory action appeared to have a decreasing tendency with the elapse of incubation time.
    PRELIMINARY OBSERVATION ON THE DETECTION OF SCHISTOSOME CIRCULATING ANTIGEN AND THE IMMUNOCOMPLEX BY DOT ELISA
    1994, 12(2):  142-143. 
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    Serum samples were collected from normal rabbits,normal persons, Schistosoma japo nicum infected rabbits and human cases respectively,and were treated by TCA precipitation and PEG precipitation,and subsequently detected by dot ELISA.The results showed that both the supernatants of TCA treated antisera from infected human cases and rabbits and the precipitants of PEG treated antisera of the same origins showed positive reactions,while no positive reactions were found for sera from normal rabbits and normal persons.It was exhi bited that dot ELISA detected not only the circulating antigen of schistosomes but also the immunocomplex.
    IN VITRO SENSITIVITY OF PLASMODIUM FALCIPARUM TO MEFLOQUINE, QUININE, AMODIAQUINE, CHLOROQUINE, PYRONARIDINE AND SULFADOXINE/PYRIMETHAMINE IN SOUTH YUNNAN 
    1994, 12(2):  144-146. 
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    The sensitivity of P.falciparum to mefloquine,quinine,amodiaquine,chloroquine,pyronaridine,and sulfadoxine/pyrimethamine(MEF,QNN,AMO,CHL,PYR,S/P)was tested using in vitro microtest in south Yunnan Province in 1992.The ID 50 were 46,480,52,150,15 and 2×10 4/25×10 2 nmol/L,respectively;ID 95 were 160,1 536,292,680,60 and 24×10 5/ 3× 10 5 nmol/L,respectively.All the isolates(30) were sensitive to MEF and QNN;100%(30/30)and 96.7%(29/30) isolates were found to be resistant to AMO and CHL,the cross resistance rate was 100% to the two drugs.One of 5 isolates exhibited resistance to PYR.The results suggest that the P.falciparum was susceptible to MEF and QNN,but was resistant to AMO,CHL and S/P. CHL resistant P.falciparum showed a marked cross resistance to AMO,but not to MEF,QNN,and S/P;AMO resistant P.falciparum exhibited cross resistance to CHL,but not to MEF,QNN,and S/P.
    HEPATIC ULTRASONOGRAPHIC IMAGING AND SERUM AMINO ACID LEVELS IN CURED SCHISTOSOMIASIS CASES
    1994, 12(2):  147-150. 
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    Ultrasonography(B mode)of liver and serum amino acid levels were studied in 15 cases of schistosomiasis japonica who had been cured for more than 5-15 years.Typical B ultraso nic images of hepatic fibrosis due to schistosomiasis were found in all patients which could be classified into three types,namely,the spotty type(Ⅰ),the tortoise shell type(Ⅱ) and the net patchy type(Ⅲ)according to the patterns of echogenic bands.Moderate to marked echogenic thickening of portal vein wall,and dilation of portal and splenic veins were revealed in patients with type Ⅱ and Ⅲ images. Free amino acids in sera were determined by using HPLC AAAS.The result revealed that the concentration of 8 nonessential and 3 essential amino acids(threonine,valine,tryptophan)were significantly decreased in the 15 patients.The ratio of aromatic to aliphatic amino acid was markedly elevated in 3 patients of type Ⅲ.The serum amino acid imbalance was at tributed to the severe hepatic damage and impaired liver function.
    STUDIES ON THE EFFICACY OF ALBENDAZOLE CANDY FOR TREATMENT OF INTESTINAL NEMATODE INFECTIONS
    1994, 12(2):  151-153. 
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    A single dose of albendazole(candy) 100 mg and 100 mg qd for two days was given respectively to 135 and 321 children who were infected with Enterobius vermicularis .All cases were cured 3wk later according to perianal tape examination.The same drug of 150 mg qd and 200 mg qd for two days was administered respectively to residents with single or multi infections of Ascaris ,hookworm and Trichuris ;the egg negative conversion rates of the two dosages were revealed to be 99.4% (466/ 469) and 99.8% (487/ 488) for Ascaris infection, 96.8% (91/ 94) and 94.3% (99/ 105) for hookworm infection,and 53.4% (228/ 427) and 76.3% (370/ 486) for Triehuris infection.The maximal numbers of worm expelled were 100 on d 2-3 ,88 on d 3-5 and 588 on d 2-4 for Enterobius,Ascaris and hookworm,respectively;whereas the discharged Trichuris were scarce,merely 4.3 in average. The results exhibited promising efficacy of the drug on Enterobius,Ascaris and hookworm infections,but not so on trichuriasis.