Table of Content

30 November 1991, Volume 9 Issue 4

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PREPARATION AND DIAGNOSTIC APPLICATION OF MONOCLONAL ANTIBODIES AGAINST SCHISTOSMA JAPONICUM

1991, 9(4):  254-257. 

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The present paper reported on an anti-CCA monoclonal antibody, McAb-ⅢD 10, which could be used in determinations of both parasite-oriented circulating antigens and specific anti-CCA antibodies. The established competitive ELISA (C-ELISA) using McAb-ⅢD 10 to detect schistospme-antibodies showed high sensitivity and specificity in the diagnosis of schistosomiasis with few cross-reactions. In field trials, coincident rates in 3 separate batches of serum samples when subjected to double-blind detections were obtained. A total of 1 915 serum samples had been determined by C-ELISA,among them 113 acute cases achieved a 100% positive rate, 765 chronic and 25 late cases showed 96.3% and 72% positive respectively. 70% of the 66 cured schistosomiasis cases turned to be negative. None of the 750 normal individuals showed positive reactions. No cross reaction was found in 27 sera from hydatidosis, whereas 1 and 2 positive reactions were found in 43 paragonimiasis sera and 126 clonorchiasis sera respectively. The established McAb-ⅢD 10 involved Dot-ELISA was found of value in the assessment of effective chemotherapy and showed a high negative conversion rate of 97.9% in 48 cured schistosomiasis patients. In 16 experimentally infected rabbits, 12 became negative in Dot-ELISA determinations at the 8th week post treatment, and the remaining 4 treated ones, the titer as well as the reaction intensity were also found reduced. A good coincidence rate was also found between C-ELISA and Dot-ELISA, their detection results may be complementary each other.

ERYTHROCYTIC SCHIZOGONY OF PLASMODIUM VIVAX UNDER VARIOUS CONDITIONS OF IN VITRO CULTIVATION

1991, 9(4):  258-260. 

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This study was performed to observe the erythrocytic schizogony of P. vivax under several culture conditions in vitro. Five experimental groups included: 1) static cultivation in candle jar; 2) static cultivation in candle jar with candle relightened; 3) static cultivation under low oxygen tension (5-10%); 4) deep suspension cultivation in test tubes with cotton plunger; 5) deep suspension cultivation in closed test tubes with screw cap. Two different isolates of P. vivax collected from Jingshan County and Zaoyang County were used. Cultivation was initiated with two methods, i.e. direct inoculation from fresh patient blood with malaria parasites and retrieval cultivation from freezing malaria parasite blood. The suspension cultivation in test tube with cotton plunger could not support the schizogony of P. vivax, while other groups could at least complete two schizogony cycles. The best result was obtained with static cultivation under low oxygen tension, the growth of parasites appeared to be more normal. The results showed that cultivation of P. vivax under a low oxygen concentration of 5-10% is preferred and the selection of isolates of P. vivax might be important in in vitro cultivation.

THE ESTABLISHMENT AND CHARACTERIZATION OF THE ANTI-IDIOTYPIC MONOCLONAL ANTIBODY-NP3O OF SCHISTOSOMA JAPONICUM

1991, 9(4):  261-264. 

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A hybridoma cell line secreting an IgM monoclonal antibody designated NP30 was ob- tained from a fusion of SP2/O and spleen cells of a BALB/c mouse chronically infected with schistosoma japonicum for one and a half year and identified by screening with immunized rabbit sera against gut-associated antigen (GAA) and soluble egg antigen (SEA) of S. japonicum, indicating that the NP30 was an anti-anti-antigen or anti-antibody. NP30 was further determined to be an anti-idiotypic antibody (anti-id) which was serologically and functionally identical to GAA, so that it could be portraied as the internal image of GAA, which might have the potential to be used as an antigenic reagent in immunodiagnostic assays, of schistosomiasis japonica.

DETECTION OF CIRCULATING MEMBRANE ANTIGEN OF SCHISTOSOME IN SCHISTOSOMIASIS BY DOT-ELISA AND IDIOTYPE/ANTIIDIOTYPE INTERACTION INHIBITION TEST

1991, 9(4):  265-268. 

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A monoclonal antibody (designated 8SE) against membrane antigen of adult Schistosomo japonicum labeled with HRP was used in dot-ELISA (direct method) to detect schistosomal circulating membrane antigen. Sera from 48 parasitologically confirmed schistosomiasis cases were tested, 39(81.396) were positive. No false positive reaction was found in sera from 24 healthy controls. No cross reaction was detected in sera from clonorchiasis or paragoni-miasis in 18 cases each. This results suggest that circulating membrane antigen does exist in patients with schistosomiasis and it might be used as a complementary method for diagnosis of schistosomiasis. A preliminary result of idiotype/antiidiotype interaction inhibition reaction for detecting circulating membrane antigen was also presented.

CLONING OF THE SEQUENCES OF KINETOPLAST DNA SPECIFIC TO LEISHMANIA DONOVAN I SPECIES AND STRAINS

1991, 9(4):  269-273. 

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To identify the heterogeneous DNA sequences in the kinetoplast DNA (kDNA) mini-circles, we digested the kDNA from various species and isolates of Leishmania into fragments with several restriction endonucleases and those fragments were southern hybridized with whole kDNA. By this test, the AluI fragments were shown to possess the species-and strain-specific sequences. We inserted these kDNA (from L. d. Sichuan human isolate) fragments into Smal site of plasmid pUC 18. The blunt ligation reaction was carried out with T4 DNA ligase supplemented by T4 RNA ligase. Tens of recombinants were obtained and at least 16 recombinants were shown to have the sequences of kDNA by colony hybridization with whole kDNA. The inserted kDNA sequences can be cut off from vector by Bam HI and EcoR I digestion. The recombinant DNA had no homolgous sequences with human genomic DNA, Leptospiral DNA, Romanomermis DNA and L. donovani genomic DNA. The clone pLKl-14, which is the smallest one among all the clones obtained, onlyhybridized to the L.d. Sichuan human isolate from which it was originated. When pLKl-14was digested by BamHI and EcoRI, a 180bp fragment comprising about 20 bp of pUC 18sequence, could be produced which corresponded to the 120±30 bp fragment of kDNA digested by Alul and was shown to be isolate specific. The clone of pLKl-10 hybridized to L. donovani isolates from hill and desert foci, might be used as a specific probe in the distinction of L.d. isolates from hill foci and plain foci. The clones pLKl-1, pLKl-2, and pLKl-15 were present in all isolates of visceral Leishmania but not in L. major and lizard Leishmania tested. These sequences might be used as specific probes in the diagnosis of visceral leishmaniasis and might be useful in epidemiological studies for identification of vectors and reservoirs.

CLINICO-EPIDEMIOLOGICAL INVESTIGATION OF SCHISTOSOME-INDUCED HEPATOSPLENOMEGALY: A COMMUNITY-BASED STUDY IN JISHAN, XINJIAN COUNTY, JIANGXI

1991, 9(4):  274-277. 

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Since the primary objective of mass chemotherapy in schistosomiasis control is reduction of schistosome-induced morbidity, it would be reasonable to assess the impact of a control program on the morbidity in a given population by investigating hepato-splenomegaly associated with schistosomiasis in a schistosomiasis endemic area. In this paper, the authors described the relationship between the prevalence, intensity and morbidity of Schistosoma japonicum infection in terms of stool egg count and ultrasonographically detectable hepatosp-lenomegaly in a community-based study. It was found that the epidemiological pattern of the infection in this study community was quite different from our usual understanding, that is, the prevalence remained relatively high (39.9%) when the intensity became lower. This unusual pattern might be resulted from intermittent ana sporadic chemotherapy associated with frequent exposure of people to the infection in an area of high transmission. It was surprised to note that despite the prazequantel treatment carried out over the past years, the prevalence of hepatosplenomegaly induced by schistosomiasis in this community was still very high, suggesting that intermittent and sporadic chemotherapy might render little impact on schistosomiasis-induced morbidity. The investigation also showed that ultrasonography was a sensitive tool for assessing morbidity associated with schistosome infection.

SEROLOGICAL CROSS REACTION BETWEEN SCHISTOSOMIASIS JAPONICA AND PAGUMOGONIMIASIS SKRJABINI

1991, 9(4):  278-280. 

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The sera from 25 patients with acute, 36 patients with chronic schistosomiasis japonica and 68 patients with pagumogonimiasis skrjabini were tested against SEA, schistosome egg, Paragonimus adult antigen (PAA) and Paragonimus metacercaria using different tests including biotin-avidin system (BAS), ELISA, circumoval precipitate reaction (COPT) and metacercaria membrane reaction (MCMR). Results showed: 1. The specific reactions using BAS and ELISA were (1) both 100% in acute schistpsomiasis patients (ASP), (2) 100% and 97.2% respectively in chronic schistosomiasis patients (CSP) and (3) both were 98.5% in paragonimiasis patients (PP). 2. The cross reactions using BAS, ELISA and MCMR were (1) 76%, 72% and 20% respectively in ASP, (2) 27.8%, 22.2% and 19.4% respectively in CSP. 3. The cross reactions using BAS, ELISA and COPT were 26.5%, 30.9% and 8.8% respectively in PP. 4. The frequency of cross reaction was related to the sensitivity of the test used. 5. The frequency of cross reaction in ASP was remarkably lower with MCMR than with BAS or ELISA. 6. The frequency of cross reaction in CSP was related to the intensity of infection. 7. We suggest that more than one test should be carried: out in patients who showed cross reaction to a single test, then the frequency of cross: reaction would decline.

ULTRASTRUCTURAL STUDY ON THE VITELLINE CELLS OF PAGUMOGONIMUS SKRJABINI

1991, 9(4):  281-283. 

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This paper reported on the ultrastructure of the developing vitelline cells of Pagumo-gonimus skrjabini by using transmission electron microscope. The vitelline glands were isolated under dissecting microscope from the adult worm of P. skrjabini before 2.5% glutaraldehyde fixation.Four developmental stages of the vitelline cell were described. In stage 1. the nucleus contained many heterochoromatins. The mitochondria, ribosomal complex,rough endoplasmic reticulum and glycogen granules were observed in the cytoplasm. In stage 2. the typical characteristic of the developing cell was the grouping together of the vitelline globules. The junctional complexes were seen between the vitelline cells. In stage 3, that represented the initial stage of vitelline droplet formation, the vitelline cells were filled with rough endoplasmic reticulum. The immature vitelline cells included stage 1-3. In stage 4. the mature cells were divided into two phases. In the early phase of stage 4, the cytoplasm contained abundant glycogen granules, lipid droplets and a lot of ribosomal complexes and rough endoplasmic reticulum,while in the later phase part of the perinuclear cisterna showed vacuolization. The cytoplasm contained a large number of glycogen granules and the vitelline droplets were distributed on the inner border of the cell. In the developing egg, the vitelline granules adhered to the egg-shell.

INTERVAL DIVISION, FORECASTING AND DECLINE TENDENCY ESTIMATION MODEL OF MALARIA INCIDENCE IN XUZHOU CITY

1991, 9(4):  284-286. 

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This paper uses Grey Model to devide the malaria incidence of 1956-1986 into interval in Xuzhou City. The Grey forecasting GM(1,1) model is used to calculate the malaria incidence in 1987 and 1988. The accuracy of forecasting is 94.83% and 82.44% respectively. Furthermore, the Grey Verhulst model is used to fit the malaria incidence for the study of the declining tendency of vivax malaria incidence. Based on the fitting models, the decline tendency estimation models of vivax malaria are worked out.

DETECTION OF PLASMODIVM FALCIPARVM INFECTION IN HUMANS BY (DIG)-DNA PROBE

1991, 9(4):  287-289. 

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In this study, we used Digoxigenin, (Dig)-11-dUTP, in the Genius?kit purchased from Boehringer Mannheim to label the Plasmodium falciparum (P.f.) DNA fragment by random primed labelling method. The P. falciparum DNA fragment was isolated from the recombinant clone pPF14 by enzyme digestion and agarose electrophoresis. This probe is designated pPF14-F-Dig.Purified P.f. DNA and blood samples from malaria patients in Hainan Province of China were detected by the probe in dot-blot hybridization. Thirty-eight patients with falciparum malaria, three with falciparum malaria and vivax malaria and four with vivax malaria were detected with the pPF14-F-Dig probe.The results showed that the pPF14-F-Dig probe could detect at least 40 pg of P.f. DNA and a parasitemia of 0.005 percent. Out of 38 detected P.f.patients' bloot samples,33 hybridized positively with this probe.The positivity rate was 86.8%. Twenty-one normal bloot samples all showed negative. In addition, 2 of 3 samples with P. f. and P. v. were positive. Three of 4 samples with P.v. were negative and one was doubtful. The preliminary result of this study indicates that the pPF14-F-Dig probe could detect the patients of falciparum malaria with good sensitivity and specificity, and that this probe can be used to detect the patients with P. falciparum in Hainan Province.

STUDIES ON STRAIN DIFFERENCES OF SCHISTOSOMA JAPONICUM IN THE MAINLAND OF CHINA VI. ANALYSIS WITH MULTILOCUS ENZYME ELECTROPHORESIS

1991, 9(4):  290-292. 

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Five different field-collected isolates (i.e., Anhui, Hubei, Guangxi, Sichuan and Yunnan) of Schistosoma japonicum from the mainland of China were compared by means of multi-locus enzyme electrophoresis in polyacrylamide gels of individual male worm extracts. The enzyme systems used in the study included GDH, G6PD, LDH, MDH, PGI, PGM and SOD. Of 9 loci examined in the 7 isozyme systems, 4 were found to be polymorphic. These were LDH-1, LDH-2, MDH and PGM, the remainder of GDH, G6PD-1, G6PD-2, PGI and SOD being monomorphic. Our results indicated that the genetic polymorphism occurring in the natural population of different isolates of S. japonicum in the mainland of China. The allele frequencies at polymorphic loci coding for enzyme as well as the genetic dentity and genetic distance in diffetent isolates of S. japonicum will be reported elsewhere.

STUDIES ON ASSESSMENT OF INTENSITY OF INFECTION FOR SCHISTOSOMA JAPONICVM IN CATTLE

1991, 9(4):  293-297. 

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In order to carry out quantitative epidemiological survey of animal schistosomiasis, a sensitive egg-counting method was studied both in the laboratory and in the field.Fecal material was filtered by a copper gauze (60 meshes/inch) into a nylon-tissue bag with 2 layers, 150 and 260 meshes/inch respectively. Thin smears were made from the residue in the bag.The mean recovery rate of mature schistosome eggs by previously adding 100 eggs to 5g of feces from noninfected cattle was 67.5%, ranging from 53 to 81%.To compare the egg-concentration thin smear method mentioned above,with egg-concentration thick smear method after hyalinization, various numbers of eggs, namely, 25, 50 and 100, were added to 5g of feces. The mean recovery rates of fecal eggs were 57.6%, 57.6% and 69.1% respectively, for the thin smear method and 3.2%, 13.8% and 19.5% for the thick smear method. The coefficients of variability in the former were from 12.9% to 17.4% and in the latter, from 30.7% to 98.8%.The total number of schistosome eggs per 5g of feces found in 34 egg-positive cattle in the field were 434 and 178 by the thin smear method and the thick smear method, respectively.A correlation analysis performed on the numbers of detected eggs in a total volume of fecal sediment and from half a volume of fecal sediment multiplied by two, showed a correlation coeffecient of 0.98 (P0.01), indicating that half a volume of fecal sediment may be used instead of a whole volume. The value of EPG, X, can be calculated from 5g of feces by applying the following formula:X(EPG)=total number of eggs in 6 slides × 2/5The adjustment value to estimate the true value of EPG from the detected one is Y= 1.1 + 1.4X.The results indicated that the egg-concentration thin smear method was significently better than the thick smear method in terms of sensitivity and reliability, so the former was considered to be useful in field epidemiological survey for animal schistosomiasis.