Table of Content

31 May 1992, Volume 10 Issue 2

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CHEMICAL SYNTHESIS AND CLONING OF PLASMODIUM FALCIPARUM 45 PEPTIDE ANTIGEN GENE

1992, 10(2):  82-85. 

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We have synthesized a 162 bp gene of human Plasmodium falciparum hybrid peptide antigen by the solid-phase phosphoramidite method with ABI 381A DNA synthesizer.The gene encodes three fragments of the relative molecules 83kDa,55kDa and 35kDa merozoite-specific proteins and two CS repeats or four peptides.The gene with the designed two cohesive ends was divided into 8 fragments to be synthesized.All synthetic fragments were annealed and ligated with T4 DNA ligase to form double DNA chain.This synthetic gene was recombined with P-Blue script as vector and transformed into E.coli JM109.The positive recombinants were screened out by dot hybridization and enzyme analysis.The DNA sequence analysis showed that the synthesized human Plasmodium falciparum hybrid peptide antigen gene was identical with the designed one.(Figs.1-4)

ISOLATION OF GERMINAL CELLS FROM THE SECONDARY CYSTS OF ECHINOCOCCVS GRANULOSVS HARBORED IN MICE

1992, 10(2):  86-89. 

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The secondary cyst tissues derived from mice infected with protoscoleces of Echinococcus granulosus for 8-10 months were digested with 0.25% trypsin at 37℃ for 30 min.The separation of different cells in the remaining suspension was achieved by discontinuous gradient centrifugation.The germinal cells were washed 3 times with ice-cold HBSS,and then cultivated in the medium of RPMI 1640 supplemented with 20% calf serum.The cells were kept in an incubator at 37℃ in an atmosphere of 95% air-5% CO2.After incubation for 5-7 days,the germinal cells began to multiply accompanied by the enlargement of cells as compared with those before incubation.The surface of both isolated and/or cultured cells showed smooth appearance examined by scanning electron microscopy Immunofluorescence assay and enzyme-linked immunosorbent assay had been used for examining the specific antigenicity of the cells.The results showed that antigen components of E.granulosus were detected either on cell surface or in soluble proteins of the cells.Furthermore,120 NIH female mice were inoculated intraperitoneally with 1-5×107 cultured germinal cells and sacrificed 1-3 months after inoculation.Only 2 cystic materials had been detected in two mice.Of which,one located in the liver and the other in peritoneal cavity of the animals.Histological examination noted that the cystic materials consisted of germinal layer and cyst fluid,but no laminated layer was observed.The above mentioned evidence demonstrated that the cells isolated from the cysts of E.granulosus were germinal cells.Since the cultured germinal cell can be kept in liquid nitrogen,it would be useful for further study on the biology,immunology and biochemistry of the cells as well as for establishing an in vitro system to screen drugs against hydatid disease.(Figs.1-9)

DNA SEQUENCE OF SURFACE ANTIGEN GENE gp195 OF TWO CHINESE ISOLATES OF PLASMODIUM FALCIPARUM FROM HAINAN

1992, 10(2):  90-94. 

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Amplification of DNA sequence of surface antigen gene,block n of gp195 from 2 Chinese isolates of Plasmodium falciparum FCC 7801/HN B3 and FCC M21/HN from Hainan,China,was established by using the polymerase chain reaction.A comparison of the 154 peptide sequences of the 2 Chinese isolates showed that they were identical,but there was a difference of 10 peptides among the established 154 peptides between the two Chinese isolates and MAD 20(a Papua New Guinea isolate).The need to determine the fragments having protective effect against malarial infection,and to study the genetic basis of the antigen polymorphism of Chinese strains of Plasmodium falciparum is evident.

TREATMENT OF SOIL-TRANSMITTED HELMINTH INFECTIONS BY ANTHELMINTICS IN CURRENT USE

1992, 10(2):  95-99. 

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The efficacy of broad-spectrum anthelmintics in current use was studied in Hengshan County,Hunan Province.The vermicides under study include albendazole (400mg,single dose),mebendazole composite (mebendazole 100mg and levamizole 25mg bid×3d),oxantel pyrantel pamoate composite(pyrantel pamoate 150mg and oxcntel pamoate 150mg bid×2d),and pyrantel pamoate composite (base 10mg/kg,single dose).Therapeutic effect assessed 2 weeks after medication revealed Ascaris egg negative rates or cure rates (CR) of 97.5-100% for the former 3 regimens,and 80.9% for the latter one,while CR for hookworm infection were 95.4%,78.6-100%,96.7% and 83.3%,respectively.A follow-up survey pursued 4 weeks post treatment showed no significant difference in CR for the above regimens.Judging from CR in Trichuris trichiura infection,pyrantel pamoate composite was recommended as the drug of choice (89.3%),which was followed by mebendazole composite (64.6-83.8%) and albendazole (28.2-42.6%),whereas pyrantel pamoate was inefficacious.Obvious egg reduction rates were evidenced post application of the above drugs in trichuriasis treatment except pyrantel pamoate at single dose.

EPIDEMIOLOGICAL TREND IN LATE STAGE OF CONTROL IN MALAYAN FILARIASIS ENDEMIC AREAS WITH ANOPHELES ANTHROPOPHAGUS AS MAIN VECTOR

1992, 10(2):  100-103. 

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During 1982-1990,a longitudinal observation on prevalence trend of malayan filariasis has been made in endemic areas with An.anthropophagus as the main vector.A total of 22 795 person-times of blood examination were made,and 30 439 An.anthropophagus and 10 061 An.sinensis were dissected respectively.The microfilaraemia rate dropped from 1.0% to 0.14%,and the infection rate of An.anthropophagus decreased from 0.74% to 0.09% in 3 endemic villages,while no positive case or infected vector occurred in 5 villages where microfilaraemia cases were absent since the beginning of the study.In a cross-sectional survey,855 villages of 17 counties has been monitored for 10 years.Out of 213 934 person-times of blood examination,only 56 were positive,the average microfilaraemia rate being 0.0262%,and 94.64% of the positive had already been detected before 1986.Based on these data,it has been suggested that in endemic areas with An.anthropopha gus as main vector,when the microfilaraemia rate dropped to1% after control,there was no indication that the rate would upgrade during the survey period.A decline trend of the transmission of malayan filariasis,therefore,has been exhibited.

STUDIES ON THE STRAIN DIFFERENCES OF SCHISTOSOMA JAPONICUM IN THE MAINLAND OF CHINA VIII. IMMUNOREACTIONS IN EXPERIMENTAL ANIMALS

1992, 10(2):  104-107. 

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Observations on the immunoreactivities toward egg antigens of different experimental animals infected with Schistosoma japonicum were made,which included mouse,rat,hamster,rabbit and rhesus monkey.The cercariae applied for infection were shed by snails collected from 5 different isolates,i.e.,Anhui,Hubei,Sichuan,Yunnan and Guangxi in the mainland of China.Sera from the same sort of animals were separately pooled according to the different sources of infection mentioned above.Antibody reactivities were evaluated by COPT,LAT and ELISA.In COPT each serum sample was tested with ova from homologous and heterologous isolates,whereas soluble egg antigen merely prepared from Anhui isolate was used in LAT and ELISA.The level of antibodies detected by homologous or heterologous worm isolate antigens was compared.The results suggested that the positive antibody defection rate might not be influenced by antigens prepared from S.japonicum eggs of different isolates in the mainland of China.

MORPHOLOGICAL AND TAXONOMICAL STUDIES ON ANISAKIDAE LARVAE FOUND IN MARINE FISHES OF CHINA II. GULF OF TONG KING

1992, 10(2):  108-112. 

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A survey on Anisakidae larvae in 29 species(134 specimens) of marine fishes in the Gulf of Tong King has been carried out.Anisakidae larvae were detected in 15 out of 29 species.The detected specimens were identified as larvae of Anisakis simplex,Hysterothylacium and Pseudoterranova.The parasitization rate of Anisakis simplex larvae,the main pathogen of anisakiasis,in fishes was 30.6% (41/134),while the parasitization rates of Hysterothylacium and Pseudoterranova larvae were comparatively low.Hysterothylacium larvae China type I detected from Muraenesox clnereus and Trichiurus Tiaumela was a new record.Their morphological characteristics were summarized as follows: 1.Length 10.78-14.18mm,Width 0.25-0.38mm,the length of the esophagus is 1.14-1.73mm,intestinal cecum 0.77-1.24mm and ventricular appendage 6.27-8.40mm,extending parallelly with the intestine to the last quarter of the larva; 2.Boring tooth was present,but mucron was absent; 3.No genital anlage was observed.

OXIDATIVE PHOSPHORYLATION OF LIVER MITOCHONDRIA IN ONCOMELANIA SNAIL

1992, 10(2):  113-116. 

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Oxidative phosphorylation of liver mitochondria in Oncomelania snail was separately detected by using oxygen electrode and spectrophotometer.ADP increased oxidative reaction of liver mitochondria from 0.187 to 0.318μmol O2/mg protein·20 min.When certain substrates of citric acid cycle were added to liver mitochondria of Oncoomelania snail,we found that oxidative phosphorylation increased to 0.353-0.444μmol O2/mg protein·20 min.ATPase was detected in the liver of Oncomelania snail.The oxidative phosphorylation of mitochondria in Oncomelania snail could be markedly inhibited by DNP and molluscicide bromoacetamide,but the latter didn't show the inhibition of ATPase.(Figs.1,2)

ULTRASTRUCTURE OF ERYTHROCYTIC STAGE OF PLASMODIUM VIVAX IN HUMANS

1992, 10(2):  117-119. 

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After the merozoite entered the erythrocyte,the membrane debris in the parasitophorous vacuoles of early ring form was passed out through a narrow external aperture in erythro-cyte to the exterior.The trophozoite was oval or irregular in shape.Ingestion of host cell cytoplasm occurred cystostomally.The asexual parasite possessed acristate mitochondria and was surrounded by a single-membraned pellicle.The gametocyte possessed cristate mitochondria and was surrounded by two unit membranes.The cytoplasm of mature macrogametocytes contained many ribosomes,mitochondria and osmiophilic bodies and a small nucleus while microgametocytes contained fewer ribosomes,osmiophilic bodies and mitochondria and a large nucleus.Three characteristic morphological alterations were observed within the host cells,that is,small vesicles,cytoplasmic cleft and caveola-vesicle complex.The clefts within the cytoplasm of the host erythrocytes were present in all human malarial parasites.The small vesicles distributed all over the cytoplasm were surrounded by a unit membrane.The caveola-vesicle complex consisted of caveolae was surrounded by small vesicles and probably corresponds to a Schuffner's dot.(Figs.1-13)

THE BLOOD SCHIZONTOCIDAL EFFECTS OF PYRONARIDINE, AMODIAQUINE, MEFLOQUINE AND QINGHAOSU ON MICE INFECTED WITH PLASMODIUM BERGHBI

1992, 10(2):  120-123. 

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The asexual stages of P.berghei ANKA were completely eliminated as revealed in a "4-day suppressive test" with the daily dose of pyronaridine 12.5mg base/kg or amodiaquine 25 mg base/kg.Mefloquine 25 mg base/kg and qinghaosu 100mg/kg though exerted obvious suppressive effect,the cure rates were only 50% and 0%,respectively.In treating chloroquine-sensitive P.berghei ANKA strain pyronaridine exhibited the best therapeutic activity,which was followed by amodiaquine,mefloquine and qinghaosu.In treating moderately chloroquine-resistant P.berghei NS line the cure rate of pyronaridine 12.5mg/ kg·d×4 was 70% ,but none of the 10 infected mice from any group was cured by amodiaquine 100 mg/kg2d,mefloquine 100 mg/kg·d or qinghaosu 200 mg/kg·d.Though the latter 3 drugs showed prominent suppressive effects,parasitemia remained positive or recrudesced after dosing.We demonstrate that parasites resistant to chloroquine had cross resistance to amodiaquine,mefloquine and qinghaosu at various degrees.Amodiaquine 100 mg/kg·d,mefloquine 100mg/kg·d and qinghaosu 200 mg/kg·d exhibited no obvious suppressive activity on highly pyronaridine-resistant line of P.berghei,indicating the existence of cross resistance to pyronaridine.

OBSERVATION ON MALARIA TRANSMISSION INTENSITY AT A STONE-PIT OF THE MOUNTAIN AREA IN BAOAN COUNTY, GUANGDONG PROVINCE

1992, 10(2):  124-127. 

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Zhangs.hubu stone-pit in Baoan County is located in the mountain area.The workers' camps are 2-3 km away from the village.In a recent survey,in malaria peak season (June,July,August) An.anthropophagus accounted for 87.7% (1059/1207) of the total anopheline population and the average man-biting rate was 10.90,24.88 and 8.71 respectively.The sporozoite positive rate,the human blood index and the daily survival rate were 0.54% (3/554),1.0 and 0.803 respectively.The parasitaemia rate,the rate of gametocyte carriers,and the rate of p rsons with malaria history within 1-2 months were 45.5% (10/22),13.6% (3/22) and 50.0% (11/22),respectively.On average,the entomological inoculation rate A,B and C calculated by three methods were 0.084,0.665 and 0.215,respectively,indicating that the workers might get malaria infection if they stayed at the camp for 1.5-11.9 days.An analysis of the malaria onset time of the patients suggested that the time calculated from the inoculation rate A was closer to the real situation than those calculated from the inoculation rate B or C.The prevalence trend of malaria could be predicted through the monitoring of the entomological inoculation rate.

OBSERVATION OF ULTRASTRUCTURE AND DRUG SENSITIVITY OF IN VITRO CULTURED EXOERYTHROCYTIC FORM OF PLASMODIUM BERGHEI

1992, 10(2):  128-131. 

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The infrastructure of in vitro cultured exoerythrocytic stage (EE) of Plasmodium ber-ghei (P.b.)was observed under transmission electron microscope (TEM).The drug sensitivity of EE was also measured in vitro.The EE was cultured in monolayer host cell,fixed and embeded in situ.The ultrathin sections were prepared and examined by routine methods.The TEM pictures showed that the fine structure of in vitro cultured EE was similar with that of EE grown in rat hepatocytes in vivo,as described by Meis et al.The parasite was found within a parasitophorous vacuole,with nucleus,mitochondria,endoplasmic reticuia and Golgi apparatus were observed inside the parasite.After 24h cultivation,the EE was incubated for 48h in medium containing a serial concentrations of primaquine or chlo-roquine,then examined under light microscope.The percentage of abnormal parasites was calculated.The preliminary results showed that the sensitivity of cultured P.b.EE to primaquine and chloroquine was significantly different.At the same concentration of 1×10-5 mol/L,the percentage of abnormal parasites was 38.5±3.9% and 5.7±1.9% ,respectively These results demonstrated that the in vitro cultivation system of P.b.EE would have potential utility in antimalarial drug research.(Figs.1-6)

THE FIRST RECORD OF HUMAN NATURAL INFECTION OF ECHINOCHASMUS LILIPUTANUS

1992, 10(2):  132-135. 

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Human natural infection with Echinochasmus Hliputanus was found for the first time from the inhabitants of Hexim county of Anhui Province in Spring,1991.Sixty worms collected from 3 infected rersons were studied morphologically.The adult worms are leaf-sh-aped,1519.4-2056.3×466.4-564.0μm in size.The width of the collar is 235.8-297.3μm.A row of 24 collar spines with size of 22.5-35.6×8.8-10.6 Mm is arranged on the collar symmetrically with dorsal and ventral interruption.The oral sucker is terminal,107.6-148.6×102.5-148.6μm.The length of the prepharynx is 25.6-66.7μm,the pharynx,97.3-127.8 Mm and the oesophagus 117.9-205.0 Mm.The caeca 764.3-1 248.8×21.0-39.0μm extends to the distal end of the body.The acetabulum is anterior to the middle level of the body,205.0-240.9×205.0-235.8μm in size.Two testes,situated in the posterior one third of the body,is slightly oval,in tandem; the anterior one is 133.3-199.9×199.9-256.4μm in size,and the posterior one,178.9-251.3×164.0-246.0μm.The Cirrus pouch is kidney-shaped,211.5-248.5×112.8-194.8μm in size,located between the bifurcation of the intestine and the ecetabulum,containing the seminal vesicle and cirrus.The ovary is oval in shape,71.8-92.3×76.8-97.4μm in size,situated in the middle of the body.The vitellaria are distributed on each side from the acetabulum to subterminal,consisting of many follicles.The uterus is short,convoluted between the anterior testis and the acetabulum,containing 0-6 eggs.The operculate eggs with thin shell are elliptical,yellowish,95.1-116.2×54.3-72.8 Mm in size.We also verified that pyquiton was effective against Echinochasmus Hliputanus.(Figs.1-4)

STUDIES ON ANTIGEMC HOMOLOGY BETWEEN SCHISTOSOMA JAPONICUM AND ONCOMELANIA HUPENSIS HUPENSIS BY INDIRECT IMMUNOPEROXIDASE TECHNIQUE

1992, 10(2):  136-138. 

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Adult Schistosoma japonicum worms and Oncomelania hupensis hupensis tissue sections were employed as antigens in the studies of anti-O.h.hupensis rabbit serum and anti-S.japonicum adult worm rabbit serum by indirect immunaperoxidase assay.The result indicated that the most prominent peroxidase reaction was presented in the adult worm sections and the head-foot and liver sections of O.h,hupensis.These suggested the possible presence of antigenic homology between Schistosoma japonicum and Oncomelania hupensis hupensis.(Figs.1-7)

PREPARATION AND IDENTIFICATION OF ANTI-TRICHOMON AS VAGINALIS MONOCLONAL ANTIBODIES

1992, 10(2):  139-142. 

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Hybridomas producing monoclonal antibodies (McAb) directed against Trichomonas vaginalis have been produced by fusing NSI myeloma cells with spleen cells of BALB/c mice immunized with Trichomonas vaginalis.IFA technique was used to test the binding activity of four McAbs produced.The McAb belonged to the IgG subtypes IgGl(2A2,2A4 McAb),IgG3 (2H9 McAb) and IgG 2b (2A12 McAb).Three McAbs,designated 2A2,2A4,2A12,reacted with a surface membrane component of live Trichomonas vaginalis.One (2A12) of them produced com-plement-dependent cytolysis of the parasites.Others (2A2.2A4) produced complement-independent cytotoxicity of the parasites.2H9 McAb which reacted with the nucleus of the organisms did not agglutinate the parasites.The four McAbs which did not have cross reaction with some protozoa of Zoomastigophorea species were specific antibodies against Trichomonas vaginalis.(Figs.1-3)