A study of the environmental determinants of malaria and schistosomiasis in the Philippines using Remote Sensing and Geographic Information Systems.

Malaria and schistosomiasis are two water-related parasitic diseases affecting millions of people worldwide particularly tropical and subtropical countries. In the Philippines, malaria is found in 72 out of 78 provinces while schistosomiasis is endemic in 24 provinces. The Anopheles mosquito and the Oncomelania snail involved in the transmission of these diseases depend on certain environmental determinants that support mosquito and snail populations. This study, done for the first time in the Philippines, successfully showed how Remote Sensing (RS) and Geographical Information Systems (GIS) can be effectively used in showing how these environmental factors affect the spatial distribution of these two diseases. The study sites, i.e. the municipalities of Asuncion and Kapalong, are known endemic sites for both malaria and schistosomiasis. Georeferenced data enabled visualization of prevalence data in relation to physical maps thus facilitating assessment of disease situation in the two municipalities. RS and GIS data proved that other factors aside from climate influence the epidemiology of the diseases in the two sites. Topography and slope as main physical factors influence the vegetation cover, land use and soil type prevailing in particular areas. In addition, water sources especially irrigation networks differed in various places in the study sites in turn affecting the magnitude and distribution of malaria and schistosomiasis. Significant correlations found between the diseases and the environmental variables formed the basis for development of models to predict the disease prevalence in the two municipalities. Proximity to snail breeding sites and irrigation networks and the highly agricultural nature of the barangays were identified as the most common factors that define the high prevalence areas for schistosomiasis confirming the fact that conditions that support the snail populations will in turn favor the presence of the disease. For malaria, the predictive models included temperature, humidity, soil type, predominance of reproduction brush, presence of cultivated areas, distance from deep wells and distance from conventional water source which are in turn influenced by the factor of elevation.

Expression of an enzymatically active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione S-transferase.

The NH2-terminal amino acid sequence of the Mr 26 000 glutathione S-transferase (EC 2.5.1.18) of Schistosoma japonicum (Sj26) has been deduced by RNA and protein sequence analysis. Using this information, a bacterial plasmid has been constructed that directs the synthesis of the entire Sj26 molecule in Escherichia coli. Recombinant Sj26 exhibits glutathione S-transferase activity and can be readily purified from bacteria in a one-step procedure under non-denaturing conditions. The availability of recombinant Sj26 in essentially unlimited quantities will aid its assessment as a candidate vaccine molecule in schistosomiasis and could eventually lead to the rational design of a drug targetted on schistosome glutathione S-transferases.

Chromosomal differentiation of the Schistosoma japonicum complex.

The C-banding pattern, location of telomere sequence and chiasma frequency of four species of the Schistosoma japonicum complex were compared with those of two African species, Schistosoma mansoni and Schistosoma haematobium. In the six species, C-banding patterns of seven autosomes and the two sex chromosomes (Z and W) showed relatively species-specific and geographical (Asian and African) differences. Particularly, a plausible pathway of alteration of chromosome 2 revealed a direction from the A-chromosome to the M- chromosome in terms of rearrangements of pericentric inversion and elimination of constitutive heterochromatin (AM inversion). This chromosome change suggested hypothetically that the S. japonicum complex is the original type, and the African species represents the derived type. Moreover, the mosaic construct of the Asian and African types in Schistosoma sinensium chromosomes prompted us to propose that the species might have been formed by hybrid speciation of the genomes of Asian and African species. Localisation of telomeric repeats enabled Asian and African schistosomes to be distinguished clearly by simple terminal location and by terminal and interstitial locations, respectively. Change of chiasma frequency in the S. japonicum complex might be caused by the reduction of interstitial chiasmate (Xi) in the larger chromosomes, 1 and Z (or W), and the change seems to have progressed to Japan from South East Asia. These data enabled us to predict a tentative evolutionary pathway of schistosomes at the cytogenetic level.

Hybridoma antibody-based competitive ELISA in Schistosoma japonicum infection.

A competitive enzyme-linked immunosorbent assay (ELISA) has been developed and compared with the circumoval precipitin test (COPT) for diagnosis of schistosomiasis japonica using Philippine sera. The assay is based on the inhibition, by sera, of the binding of a penicillinase-conjugated hybridoma-derived antibody, I. 134, to a crude Schistosoma japonicum adult worm extract. A change in pH subsequent to addition of the substrate is used as the indicator system. Development of the color change in this assay is relatively slow, a fact which presumably facilitates detection of inhibition by serum. Relative to the COPT, no false positive reactions were obtained and the false negative rate was less than 10%. A wide range of inhibitory titers was obtained using sera in the competitive ELISA similar to that found in a competitive radioimmunoassay using 125I-labeled I. 134. The competitive ELISA will be of more general application for diagnosis of schistosomiasis japonica than the competitive RIA using hybridoma antibodies, and will provide more precise quantitative information than is obtainable in the COPT.

Sensitization against the parasite antigen Sj26 is not sufficient for consistent expression of resistance to Schistosoma japonicum in mice.

Mice immunized with purified antigen preparations produced in Escherichia coli and containing the glutathione S-transferase (GST) isoenzyme of Schistosoma japonicum (Sj26) can be partially resistant to infection with this parasite. Maximum resistance was approximately 50% and no protection was obtained in BALB/c mice, known low responders to Sj26. Although only Freund's complete adjuvant has been used, the data obtained indicate that satisfactory levels of resistance to S. japonicum will not be attained by vaccination with Sj26 alone. Other antigens, including the additional GST isoenzyme of S. japonicum Sj28, will probably be required to establish whether Sj26 will be an important component of a defined multivalent vaccine against schistosomiasis japonica.

Schistosome circulating anodic antigen in serum of individuals infected with Schistosoma japonicum from the Philippines before and after chemotherapy with praziquantel.

The presence of the schistosome circulating anodic antigen (CAA) in serum of patients infected with Schistosoma japonicum from The Philippines has been investigated using an enzyme-linked immunosorbent assay (ELISA). Serum samples were tested from 48 patients who excreted S. japonicum eggs, 9 individuals with a negative stool examination, and 20 controls with both a negative stool and a negative circumoval precipitin test. No false positive result was detected for the unequivocally negative controls. CAA could be demonstrated in 72.9% of the egg-excreting patients. A positive correlation between parasite burden (eggs per gram of faeces) and antigen level (CAA titre) was found (Spearman's rho = 0.48, P < 0.001, n = 48). Four of 18 sera from the egg-negative individuals were positive in the ELISA. In view of the fact that anti-worm antibodies were also detected in these 4 sera, those reactions suggest active infection not detected by stool examination. In serum from patients treated with praziquantel, a significant drop in CAA titre was seen within 5 d after treatment (Wilcoxon's chi T = -2.23, P = 0.0258, n = 21). In conclusion, the detection of CAA by ELISA in S. japonicum infection can give valuable information in both individual diagnosis and therapeutic drug monitoring, as well as in epidemiological studies or disease control programmes.

Nuclear and mitochondrial genetic markers highly conserved between Chinese and Philippine Schistosoma japonicum.

Geographical isolates of S. japonicum, and particularly isolates from China and the Philippines, were examined at the molecular level for genetic divergence. Sequences from both nuclear and mitochondrial genomes were selected as markers of evolutionary divergence and S. mekongi and S. mansoni were included in the study for comparison purposes. Restriction fragment length polymorphism (RFLP) and PCR-RFLP analysis of the rDNA repeat unit and sequence analysis of the second internal transcribed spacer region (ITS2) within the rDNA repeat and the cytochrome c oxidase I (COI) gene of the mitochondrial genome were performed. No intra-specific variation in S. japonicum was found in the rDNA repeat and only very slight variation was detected within the COI sequence. A survey of the entire genome, using random amplified polymorphic DNA (RAPD) analysis, again showed that Chinese and Philippine S. japonicum are remarkably similar at the DNA sequence level. We were thus unable to obtain direct molecular evidence in support of previous findings, particularly those based on isoenzyme analysis, that a very high level of intra-specific variation exists in S. japonicum.

Monoclonal antibodies reacting with Schistosoma japonicum eggs and their target epitopes.

Ten monoclonal antibodies (McAbs) raised to Schistosoma japonicum eggs could be assigned using several serological and immunochemical techniques to 3 groups. The McAbs, termed A, B and C-McAbs, apparently recognize carbohydrate epitopes that can be located on the same antigen molecule. The antibodies, generally of IgM isotype, are idiotypically related. They are distinct from another IgM McAb (Group D-McAb) the carbohydrate target epitope of which can also be associated with the epitopes of A, B and C-McAbs. The McAbs produce large vacuolated bleb reactions in the circumoval precipitin test (COPT) and target epitopes have different representations in various life cycle stages such as immature and mature eggs, male and female worms (including S. mansoni). Antigens affinity purified on columns containing A, B, C and D-McAbs stimulate proliferation of T cells from egg-sensitized mice and elicit DTH reactions in such mice. This raises the possibility that the target antigens of these carbohydrate-reactive monoclonal antibodies are immunopathologic and involved in egg-induced granuloma formation.

Effects of induction of anti-embryonation immunity on liver granulomas, spleen weight and portal pressure in mice infected with Schistosoma japonicum.

BALB/c mice sensitized with injections of viable immature Schistosoma japonicum eggs had significantly fewer and smaller granulomas in the liver, lower portal pressure and smaller spleens at D + 75 of infection compared to similarly infected unsensitized controls. The portal pressure and spleen weights of the mice sensitized with immature eggs were not different from uninfected unsensitized mice of similar ages at D + 75 of infection. The results strongly support our hypothesis that it should be possible to prevent serious hepatosplenic disease in schistosomiasis japonica by vaccination to induce anti-embryonation immunity.

Schistosoma mansoni and S. japonicum worm numbers in 129/J mice of two types and dominance of susceptibility in F1 hybrids.

In a study on the genetics of resistance to schistosomiasis in WEHI 129/J mice, susceptibility to either Schistosoma mansoni or Schistosoma japonicum was shown to be unequivocally dominant in F1 hybrid crosses between genetically resistant WEHI 129/J and susceptible BALB/c mice. The operation of only 1 or 2 genes in the expression of resistance to S. mansoni was suggested by backcross analysis. Thus, approximately 25% of (BALB/c x WEHI 129/J) F1 x WEHI 129/J mice were resistant to S. mansoni infection, whereas resistance was manifest in approximately 50% of WEHI 129/J mice. The data are consistent with resistance being controlled by 1 recessive gene having 50% penetrance. We also report that 129/J mice obtained directly from the Jackson Laboratories (Bar Harbor, Maine) (designated JAX 129/J), differ from locally bred WEHI 129/J in being entirely susceptible to S. mansoni infection. However, both WEHI 129/J and JAX 129/J are relatively resistant to S. japonicum infection.

A preliminary examination of the major integral membrane protein antigens of Schistosoma japonicum and Schistosoma mansoni adult worms for glycosyl-phosphatidylinositol membrane anchors.

Integral membrane protein (IMP) antigens isolated from S. japonicum and S. mansoni adult worms using Triton X-114 phase partitioning were treated with phosphatidylinositol-specific phospholipase C (piPLC). Following piPLC treatment, only one IMP antigen of 58 kDa from each species was released from the hydrophobic fraction and remained soluble in the absence of detergent. An additional 23 kDa antigen was identified following piPLC treatment of S. japonicum IMP's. This molecule has been previously characterized as an important species specific immunodiagnostic antigen. Alkaline phosphatase activity was observed in both the detergent and aqueous phases following treatment with piPLC but only in the hydrophobic fraction of the controls. These data suggest that only a small number of IMP antigens from both S. japonicum and S. mansoni adult worms possess glycosyl-phosphatidylinositol (GPI) lipid membrane anchors in a form which can be hydrolysed by a heterologous piPLC.

Resistance to reinfection with Schistosoma japonicum in the mouse.

BALB/c and outbred mice infected with a Philippine isolate of Schistosoma japonicum for 50 to 60 days expressed strong resistance to reinfection. The extent of this reinfection resistance ranged from 72 to 93% in 5 experiments (mean = 80% resistance) as determined by numbers of immature worms recovered from already infected and age- and sex-matched challenge control mice exposed 20 days previously to cercariae. Determination of numbers of recoverable worms from (the initial) infection suggest that adult worms are lost progressively during the period in which impressive resistance to reinfection is demonstrable. An important unresolved question is whether loss of adult worms is related in any way to expression of resistance to reinfection. Some indirect evidence indicates that the major component of reinfection resistance is expressed prior to day 4 of challenge infection. This evidence derives from analysis of lung petechiae which, in a primary infection, have been shown to provide an indication of number of adult worms which can be detected subsequently (e.g. at 30-40 days of infection). Although anti-parasite immune response have not yet been shown to be responsible for this apparent concomitant immunity, the magnitude of resistance to reinfection in the S. japonicum/mouse system should facilitate identification of any immunological effector mechanisms involved.

Attempts to standardize the circumoval precipitin test (copt) for Schistosomiasis japonica.

The circumoval precipition test (COPT) is a simple and inexpensive immunodiagnostic test for schistosomiasis japonica which, in the Philippines, has high sensitivity and specificity. Lack of standardization does, however, increase the variability of the test. Parameters which influence the COPT have been examined using large numbers of sera from known S. japonicum infected individuals. In this series of experiments, optimal conditions were determined to be as follows using 2 drops of neat serum and incubation at 37 degrees C in a sealed slide chamber; - approximately 100 eggs from 55 or 60 days infected rabbits for a 24 to 48 hour incubation period. COP reactions (i.e. precipitates associated with eggs) were much less obvious when either immature eggs or eggs obtained from long-term infected rabbits were used. The results emphasize the prime importance of the source of Schistosoma japonicum eggs in the performance of a standardized COPT.

Schistosoma japonicum: the modulation of lung granuloma and inhibition of egg maturation in mice by human sera.

Human sera taken from patients with chronic schistosomiasis japonica have been demonstrated to have two effects on mice. Sera from those patients reduced the size of granuloma in mice sensitised for accelerated granuloma formation to eggs entrapped in the lungs of mice injected with the sera shortly before and at day 2 after intravenous egg challenge. The sera with this effect on the mouse lung granuloma models caused large segmented precipitates in the optimised circumoval precipitin test (COPT). Such sera also reduced the rate at which eggs matured in the liver and intestines of mice infected with S. japonicum. The results strongly support our postulate that a major cause of granuloma modulation in cases of chronic schistosomiasis japonica is antiembryonation immunity and that mice provide useful models for the analysis of our postulate. Identification of egg antigens responsible for the anti-embryonation effect should facilitate progress towards the development of a vaccine against granulomatous disease.

Evidence of anti-embryonation immunity and egg destruction in mice sensitized with immature eggs of Schistosoma japonicum.

BALB/c mice sensitized by repeated injections of immature eggs of the trematode worm, Schistosoma japonicum, were challenged with low numbers of cercariae and evidence was sought for inhibition of embryonation by examination of eggs in livers and intestines at days 40 - 42 of infection. In contrast to the situation in unsensitized control mice, a greater proportion of dead eggs was noted in tissues of many of egg-sensitized mice. There was also a decrease in the proportion of mature eggs relative to control mice. A substantial number of egg - sensitized mice contained no eggs in the liver though eggs were readily detected in their intestinal walls. The data support the concept that immune effector mechanisms act on eggs in a manner that prevents their full development into a miracidium and thus a rich source of immunopathologic antigens.