Table of Content

31 December 1998, Volume 16 Issue 6

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论著

IMMUNIZATION OF MICE WITH NATIVE TROPOMYOSINS FROM SCHISTOSOMA JAPONICUM AND ONCOMELANIA HUPENSIS

CaoJianping;LiuShuxian

1998, 16(6):  401-405. 

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AIM: To study the protection against cercariae challenge in mice immunized with native tropomyosin (TM ) antigens from Schistosoma japonicum (Chinese strain) and Oncomelania hupensis. METHODS: The native TM antigens were extracted from adult worms of S. japonicum (SjcTM ) and head-pad of O. hupensis (O hTM ) , the purity and the molecular weight of these antigens were analysed by SDS-PAGE. Each C57BL/6 female mouse was immunized subcutaneously by injection of 5 μg SjcTM o r 30 μg O hTM emulsified in an equal volume of Freud’s adjuvant for three times. Control mice were in jected only with adjuvant. All the mice were artificially challenged with cercariae of S. japonicum at five days after final injection. Six weeks after challenge, the mice were perfused, the worms and the eggs collected from the livers and the intestines of mice were counted. RESULTS: The prepared SjcTM and O hTM antigens were shown to be pure, the molecular weight being around 40 kDa. Compared with the control group, the worm reduction rates of SjcTM group and O hTM group were 21.1% and 28.2% , respectively; the egg reduction rates per worm pair in the tissues were 20.1% and 52.2% (liver) , 32.8% and 28.1% (intestine) , respectively; the matured egg reduction rates in the tissues were 25.9% and 46.8% (liver), 39.7% and 66.8% , respectively. CONCLUSION: SjcTM and O hTM antigens can be served as new candidates of vaccine against schistosomiasis japonica.

CLONING AND SEQUENCING OF THE GENE CODING THE SEXUAL STAGE ANTIGEN Pfs48/45 OF PLASMODIUM FALCIPARUM

LuoShuhong;YuXinbing;LiuYanwen;FangJianming;XuJing;LiXuerong

1998, 16(6):  406-410. 

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AIM: To express the antigen Pfs48/45 in vitro and provide an antigen for the
development of the transmission blocking vaccine. METHODS:According to the published nucleotide
sequence of Pfs48/45 of Plasmodium falciparum isolate NF54, a pair of oligonucleotides was
designed and used as primers(P1,P2). The gene encoding the gametocyte/gamete specific membrane
protein Pfs48/45 of P. falciparum isolate FCC1/HN has been amplified by using polymerase chain
reaction(PCR) technique. The PCR product was purified and directly sequenced by the dideoxynucleotide terminator method with 5’-end primer P1. At the same time, the purified PCR product was digested with BamHI and EcoRI and cloned into the plasmid pcDNA 3, then the recombinant clones were transformed into E. coli strain TG1. The recombinant plasmid pcDNA 32Pfs48/45 was screened and identified by PCR amplification and restriction analysis. RESULTS: ① The gene fragment Pfs48/45 was specifically amplified from the genomicDNA of Plasmodium falciparum isolate FCC1/HN; ②The sequence demon-strated that the 5’-end nucleotide and predicted aminoacid sequence of Pfs48/45 from FCC1/HN isolate was basically identical with that from NF54 isolate. We found that the sequence of the Pfs48/45 gene from the isolate FCC1/HN differs from the published sequence (isolate NF54) only at positions 307 and 372 (T →C). The substitution of T →C at the position of 372 generates a new restriction site Taq I. The PCR product digested by Taq I generates DNA fragment of 984 bp and 379 bp , suggesting that the PCR product is the gene encoding the transmission blocking antigen Pfs48/45; ③ The gene fragment of
Pfs48/45 was directly inserted into the BamHI and EcoRI site of plasmid pcDNA 3. CONCLUSION: The nucleotide sequence of Pfs48/4 5 of Plasmodium falciparum isolate FCC1/HN from south China was similar to that of isolate NF54. The recombinant plasmid pcDNA 3 Pfs48/45 was successfully constructed, providing a means to evaluate the role and biological function of this sexual-stage-specific protein of Pfs48/45.

CROSS PROTECTION AGAINST SCHISTOSOMA JAPONICUM INFECTION IN MICE IMMUNIZED WITH TRICHNELLA SPIRALIS MUSCLE LARVA ANTIGEN *

TianMingli;YiXingyuan;ZengXianfang;ZengQingren;PengXianchu

1998, 16(6):  411-414. 

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AIM: To explore the cross protection against S.japonicum cercaria challenge in mice
immunized with T. spiralis larva antigen. METHODS:Groups of BALB/c mice were immunized with 4
preparations of T.spiralis larva antigen, respectively.Vaccinated mice were then challenged with
30 and 100 S.japonicum cercariae,respectively.Forty five days later,all mice were sacrificed and
examined for adult worm burden,liver and stool egg count. RESULTS: All groups of mice immunized
with 4 preparations of T. spiralis larvae antigen withou tadjuvant showed significant protection against S. japonicum. Among these antigen preparations, the soluble antigen (TsLSA ) presented highest protection with reductions in worm burden and liver and stool egg counts of 21.3% , 48.0% and 58.5% , respectively. When mice were immunized with TsLSA plus Freund’s complete adjuvant (FCA) or with double-dose of the antigen, the worm reduction rate was increased to 29.3% and 39.6% , respectively. CONCLUSION:Different preparations of T. spiralis muscle larva antigen could induce protection against S. japonicum in mice.

CLONING AND EXPRESSION OF SCHISTOSOMA JAPONICUM EGGSHELL PROTEIN GENE

LiuLanying;YuXinbing

1998, 16(6):  415-420. 

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AIM: To construct an eukaryotic expression system pcDNA3/ESG HeLa cell and explore
the possibility of whether the eggshell protein might be used as potential targets for vaccine
development. METHODS: The eggshell protein gene(ESG)was amplified using PCR technique and
inserted into vector pcDNA3 to construct recombinant vector pcDNA3/ESG. The expression of the
eggshell protein was performed in HeLa cell line.SDS PAGE,dot ELISA and Western blotting were
used to identify the antigenicity of the expressed product. RESULTS: The amplification of ESG (618) bp was achieved and high level expression of the eggshell protein was obtained in HeLa cells and the amount of total expressed products was up to 24.4%. Dot ELISA and Western blot analysis demonstrated that the expressed protein could be recognized by sera from rabbits infected with S.japonicum and from female worm-or egg-immunized rabbits. In addition, the expressed protein could specifically stimulate proliferative response of the immunized BALB/c mouse spleen cells, the transformation rate being up to 44.57%. CONCLUSION: The eukaryotic expression system pcDNA 3/ESG was successfully established and high level expression of the eggshell protein of S.japonicum was achieved in HeLa cell line. The expressed recombinant protein exhibited immunological activities.

EFFECT OF DIHYDROQINGHAOSU ON THE DEVELOPMENT OF PLASMODIUM YOELII YOELII IN ANOPHELES STEPHENSI

ChenPeihui;TuYouyou;WangFengyun;LiFengwu;YangLan

1998, 16(6):  421-424. 

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AIM:To observe the effect of dihydro qinghaosu (DQHS) on the development of Plasmodium yoelii yoelii in Anopheles stephensi and to explore the possibility of whether DQHS has preventive effect against malaria.METHODS: Plasmodium yoelii yoelii infected mice that had been administered with a single dose of different dosages (60 mg/kg,120 mg/kg,180 mg/kg and 240 mg/kg) of DQHS were exposed to mosquitoes to suck blood .The development of malaria parasites in the mosquitoes of the control group and the treated group were observed by light microscopy (LM ) and transmission electron microscopy (TEM ). RESULTS: DQHS exhibited certain inhibitory effect on the infectivity of gametocyte. The degree of inhibition was related to the developmental stages of gametocyte and the drug dosage. Immature gametocytes were more sensitive to DQHS than the mature gametocytes. With the increases in dosage , the positive rate and the den ity of oocyst and sporozoite decreased. However, the difference in the density of sporozoite between 180 mg/kg and 240 mg/kg treated groups was in significant. TEM result showed damage of the membrane system and vacuolation in the cytoplasm of the oocysts (12- 13 d) on mosquito midgut of 60 mg/kg 16 h treated group. The oocysts in the mosquitoes continued to develop after treated with 120 mg/kg of DQHS for 16 h on 3-day-in star. There was no significant difference (P >0.05 ) in the density of oocyst and sporozoite between the control group and the treated group. CONCLUSION:DQHS can affect the infectivity of gametocyte and decrease themalaria transmission but cannot inhibit the sorogonic stage directly.

KARYOTYPE ANALYSIS OF LEISHMANIA ISOLATES IN CHINA *

WuBo;QuJingqi

1998, 16(6):  425-431. 

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AIM: To further understand the genetic variation among Leishmania species in China by karyotype analysis. METHODS: DNA karyotypes of 16 Leishmania isolates in China were analyzed by pulsed field gel electrophoresis, and 5 different species of WHO reference Leishmania isolates were used in comparison. RESULTS: Chromosomal DNAs under 2 200 kb were resolved under pulsed field gel electrophoresis conditions. All the 16 isolates, even isolates in the same endemic area exhibited different chromosomal DNA banding patterns. The size variation in the chromosomal DNAs was evident particularly in chromosomes between 225 and 850 kb. Large-sized DNAs of more than 850 kb were more conserved than smaller-sized DNAs among 16 Leishmania isolates. DNA karyotype analysis showed: ① The 14 isolates belonging to L. infantum /donovani complex presented similar but different karyotype; the DNA banding patterns of those from the same endemic area were more similar than different areas, as seen in isolates of KS22, KS23, KS26, KS27, KS28, KS29, KS213 and KS216 in Kashi region, Xingjiang and in isolates of Liu and Xu in Karamay region, Xingjiang. The 771 isolates separated from sandfles in Bachu, Xingjiang exhibited 26 DNA bands, being more than the average 19- 21 DNA bands of other isolates separated from human patie ts. ② R12 and R 20 isolates having been previously named after L. turanica and L. gerbilli exhibited much more similar karyotypes, which were obviously different from other tested isolates and the five WHO reference isolates. CONCLUSION: ① Apparent in traspecific karyotype variations among L. donovani/infantum complex isolates in China, the more similarities in DNA banding patterns, the closerrelationship between the isolates. ② Eco-geographical isolation and sandfly vector have contributed to the heterogeneity of L.infantum /L.donovani complex in China. ③ The isolates R 12 and R20 tend to belong to one species/two closely-related species.

ANALYSIS OF NUCLEAR DNA GENE TYPES OF LEISHMANIA ISOLATES FROM HILLY AND PLAIN FOCI OF CHINA

LuFangli;HuXiaosu;JingBaoqian;MaYing

1998, 16(6):  432-435. 

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AIM: To analyse the nuclear DNA (nDNA) polymorphism of Leishmania isolates from hilly and plain foci of China. METHODS: nDNA were analysed by endonuclease digestion, Southern blotting and chromosomal localization. Probes were labeled with digoxigenin. RESULTS: Using gp63 gene probe, similar hybridization bands were found to be existed between nDNAs of Leishmania donovani Jiangsu human isolate and L.d.Jeddah, also between nDNAs of L.d. Sichuan human isolate and L.infantum. Using β-tubulin gene probe, there were two similar hybridization bands existed between nDNAs of L. d. Jiangsu human isolate and L. d. Jeddah, and three similar hybridization bands existed between nDNAs of L. d. Sichuan canine isolate and L. d. Gansu canine isolate, and two similar hybridization bands existed between nDNA of L. d. Sichuan canine isolate and L. d. Wenchuan human isolate. CONCLUSION: Homology exists between L. d. Jiangsu human isolate and L. d. Jeddah from plain foci, between L. d. Sichuan human isolate from hilly foci and L. infantum, between L. d. Sichuan canine isolate and L. d. Gansu canine isolate from hilly foci. Homology
as well as differences exist between L. d. Sichuan canine isolate and L. d. Wenchuan human
isolate. Heterogeneity exists between isolates from hilly foci and plain foci.

CORRELATION BETWEEN THE DOSE AND THE ANTI TOXOPLASMA EFFECT OF ACTIVATED MOUSE MACROPHAGES INDUCED BY IFN-γ AND THE SYNERGISM BETWEEN IFN-γ AND TNF-α

ZhangAimin;YangHuizhen;YangYang;QianZhongli

1998, 16(6):  436-440. 

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AIM: To find out the dose dependence of the anti-Toxoplasma effect induced by IFN-
γ and to determine the possible synergistic activity between TNF-α and IFN-γ. METHODS: The in
vitro effect of cultivated mouse peritoneal macrophages activated by IFN-γ alone or IFN-γ
combined with different doses of TNF-α on the intracellular tachyzoites of RH strains and the
nitric oxide (NO) level in the culture medium supernatant were simultaneously determined.
RESULTS: With the increase in the dose of IFN-γ, the anti-Toxoplasma effect was augmented and the NO level was enhanced. At 24 hours after tachyzoite invasion, a significant reversed correlation was demonstrated between the NO level and the number of intracellular parasites. CONCLUSION: The anti-Toxoplasma effect of macrophages activated by IFN-γ appears to be dose-dependent and TNF-αacts synergitically with IFN-γ in the activation of macrophages. The production of reactive NO could be an important effector in the IFN-γ primed anti-Toxoplasma action.

DETECTION OF HFRSV IN EULAELAPS SHANGHAIENSIS AND ORNITHONYSSUS BACOTI BY USING IN SITU HYBRIDIZATION

WuJianwei△;MengYangchun;LiYunhe;ZhouHongfu;ZhugeHongxiang;LaiPeixin;WangJianming

1998, 16(6):  441-444. 

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AIM:To provide molecular biological evidence of transmission of hemorrhogic fever
with renal syndrome virus (HFRSV) by gamasid mites, Eulaelaps shanghaiensis and Ornithonyssus
bacoti .METHODS: Frozen sections of the gamasid mites 10 days and more than 100 days after
biting suckling mice inoculated with HFRSV were in situ hybridized with dig labelled HFRSV cDNA
probes .RESULTS: RNA was detected in frozen sections of Eulaelaps shanghaiensis , after biting
suckling mice inoculated with Hantaan (76-118) and Seoul (UR ) virus, respectively. Most of the fine granules of the virus RNA were located in the nuclei, cytoplasm and nuclear membrane of cells of brain cortex, caeca and genitalia of the mites. In situ hybridization results showed that 17 of 31 mites in Hantaan group and 10 of 23 mites in Seoul group were positive. The virus RNA was still detected in tissues of the mites on d132 for Hantaan group and on d102 for Seoul group after infection, respectively. Among 20 Ornithonyssus bacoti detected 12 were positive on d10 after biting suckling mice inoculated with Hantaan virus, and the virus RNA was mainly found in the cells of genitalia (Figs. 1- 13). CONCLUSION: Both Eulaelaps shanghaiensis and Ornithonyssus bacoti could be infected with HFRSV by biting HFRSV-positive mice . E. shanghaiensis could be infected with both Hantaan and Seoul virus, and the two types of HFRSV were found to be maintained in the mites for 132 days and 102 days, respectively. These confirmed that E. shanghaiensis and O. bacotiare suitable vectors and reservoirs of both Hantaan and Seoul virus and might play an important role in the cross transmission of the two types of HFRSV.

STUDIES ON THE EXPERIMENTAL TRANSMISSION OF RATTUS BORNE HANTAVIRUS BY ORNITHONYSSUS BACOTI

ZhugeHongxiang;MengYangchun;WuJanwei;ZhuZhiyong;LiangWiefeng;YaoPingping

1998, 16(6):  445-448. 

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AIM: To demonstrate the role of rat mite ( Ornithonyssus bacoti ) in the
transmission of Rattus borne hemorrhagic fever with renal syndrome (HFRS). METHODS: In the
transmission experiments, about 100 O bacoti per pool were isolated and placed in a jar, unfed
for 4 d at 23±1 ℃. Suckling Wistar rats inoculated with Hantavirus strain Z45 or Seoul virus
strain UR were placed in each jar for free attack by the mites for 12 hours. After 14 d the
normal suckling Wistar rats were bitten by the mites Fifteen days later, the lung tissues and sera of the infected rats were collected and detected for Hantaviral antigen by indirect fluorescent antibody technique (IFAT). For demonstration of the infection of O.bacti with Rattus-borne Hantavirus PCR technique was applied to detect Rattus-borne Hantaviral RNA. RESULTS: Sukling Wistar rats inoculted Hantavirus strain Z45 or Seoul virus strain UR were bitten by O. bacoti and then these mites were fed on 4 and 5 of normal suckling rats in each Jar, respectively. The antigens of Hantavirus strain Z45 were postive in all the lungs of the normal rats bitten by the mites, the sera titers of the rats were from 1∶10 to 1∶40. The antigens were postive in 3 of the 4 rats, the sera titers were from 1∶20 to 1∶40. Both of the viruses could be maintained in O.bacoti for 22 days. The blocking test showed when 1∶20 Hantavirus immuno sera were exposed to the lung samples and then reacted with the sera from the
patients with HFRS, all the specific fluorescence reactions of the samples were blocked,
whereas the control group including the normal rat lung tissues and sera were all negative
(Fig. 1). CONCLUSION: O.bacoti might play a role as the vector of HFRS and a reservoir host as well.

EVALUATION OF THE EFFECT OF CAUSES OF DEATH OF ADVANCED SCHISTOSOMIASIS PATIENTS ON THE LIFE SPAN OF POPULATION BY USING POTENTIAL YEARS OF LIFE LOST ANALYSIS

HuangDesheng;MaXingbao;CaiLi

1998, 16(6):  449-453. 

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AIM: To evaluate the effect of the causes of death of advanced schistosomiasis
patients on the life span of population in an area where schistosomiasis had been under control
by applying the indicators of potential years of life lost (PYLL) and rate of PYLL(PYLLR).
METHODS: Using the formula of calculation of PYLL and PYLLR,the causes of death of 487 patients
of advanced schistosomiasis in 3 previously endemic townships of Shanghai suburb . RESULTS: Of
487 cases who died between 1955 and 1995, the PYLLR of all causes of death was 256.3‰. The main causes of death were hepatic fail re ( 100.3‰) , liver carcinoma (43.4‰) and cancer of other organs (24.8‰). In the 1960s, the PYLLR of advanced schistosomiasis comlicated with hepatic failure and upper digestive tract hemorrhage was 222.2‰ and 34.5‰, the PYLLR of the complications of schistosomal liver cirrhosis, hepatic failure and hemorrhage accounted for 67.5% of the total PYLLR, being apparently higher in the non-splenectomy group than in the splenctomty group . After the schistosomiasis was under control, both PYLL and PYLLR decreased linearly. As compared with the 1960s, the PYLLR of all causes of death and the causes of death of the complications of schistosomal liver cirrhosis in the 1990s has declined by 62.9% and 83.2% , respectively, the PYLLR of upper digestive tract hemorrhage was found only 0.3‰ in the last 5 years. CONCLUSION: PYLL and PYLLR analysis could be used to quantify the effect of complications of advanced schistosomiasis and other causes of death on the life span of population.

实验报道

APPLICATION OF DOT IMMUNOGOLD FILTRATION ASSAY FOR DIAGNOSIS OF SCHISTOSOMIASIS JAPONICA

ZhangEnying;WuChenyun;ShenDakang

1998, 16(6):  454-456. 

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AIM:To quest for a simple and convenient assay for the diagnosis of
schistosomiasis. METHODS: Three monoclonal antibodies against different epitopes of antigens of
S. japonicum were used to develop a dot immunogold filtration assay for detection of
schistosomal antigen antibody reaction on the membrane during the filtration process.The results
could be read with the naked eye within a few minutes. RESULTS: The sensitivity of the SEA assay
was 16 ng/ml.In 69 serum samples from patients with chronic schistosomiasis, the positive rate and specificity of this assay were 60.8% and 95.2% , respectively. Dot-ELISA was used in detecting 137 serum samples from patients with chronic schistosomiasis, the positive rate and specificity were 54.7%and 94.6%, respectively. When sandwich-ELISA was used in detecting 118 serum samples from patients with chronic schistosomiasis, the positive rate and specificity were 61.9% and 95.7%, respectively. CONCLUSION: The sensitivity and specificity of DIFA were similar to those of ELISA, but DIFA required less time and no special equipment.

防治经验

EXPLORATION ON STATISTICAL METHOD FOR CALCULATING INTENSITY OF THE INFECTION IN RESIDENTS IN NATIONWIDE SAMPLING SURVEY OF SCHISTOSOMIASIS

WangYanan;HuaZhenghui;WuWeiping;GuoJiagang;WuXiaohua;ZhengJiang

1998, 16(6):  457-459. 

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AIM: To set up statistical methods for calculation of intensity of schistosome
infection in residents in nationwide sampling survey of schistosomiasis. METHODS: Based on the
epidemiological survey of schistosomiasis and according to the medical statistics for multiple
stratifying and clustering random sampling,the calculation method on intensity of schistosome
infection was inferred. RESULTS AND CONCLUSION: Nine formulas were worked out for the
calculation of the intensity of schistosome infection in different endemic provinces with different ecotypes. These formulas are of practical value.

IN VITRO SENSITIVITY OF PLASMODIUM FALCIPARUM TO CHLOROQUINE,PIPERAQUINE,PYRONARIDINE AND ARTESUNATE IN YUXI PREFECTURE OF YUNNAN PROVINCE

FanBo;ZhaoWei;MaXinwen;HuangZhengmei;WenYunsheng;YangJiqing;YangZongxiang

1998, 16(6):  460-462. 

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AIM:To assess the sensitivity of Plasmodium falciparum to four antimalarials.
METHODS:WHO standard in vitro microtest was used.RESULTS: The resistance rates of the malaria
parasite to chloroquine,piperaquine pyronaridine and artesunate were 85 7%, 66 7%, 38 1% and 5
0%,respectively. CONCLUSION: Among the antimalarials tested in Yuxi Prefecture,Yunnan
Province,high resistance of P.falciparum to chloroquine and piperaquine was found.The
sensitivity of P.falciparum in part of the case to pyronaridine decreased. However, most of the cases were relatively sensitive to artesunate.