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Eradication of schistosomiasis japonica in Yamanashi Prefecture was officially declared in 1996, and all surveillance and health campaign were finished by the end of 2001. Schistosomiasis control had been carried out by strong collaboration among local Government, local people and academia, thought which knowledge and experiences of the disease control were accumulated among the local people. It is 20 th anniversary of the disease eradication in Yamanashi. We planned to analyze the current situation whether the local people still keep the knowledge of schistosomiasis or not. There was no more knowledge kept in the middle school students, to whom the local Government did not educate about schistosomiasis. Among adult individuals, elderly people who have experienced endemic condition still know about it at almost comparable level as in the past. However, younger people who learned it in the primary school but no experience of the disease transmission started losing the knowledge. Those situations of the adult local people were compared with the veterinarian group. It was confirmed that the veterinarian group had correct and proper knowledge of schistosomiasis japonica not only in elderly groups, but also in younger age group. Considering that methods for control and prevention of infectious diseases are common to some extent, their knowledge and experiences would be applicable for other infectious diseases in future. Therefore, the knowledge would be worth keeping in the local people. Taken together, individuals with specialty, such as veterinarians, are expected to play roles in public health for promotion of health and welfare.

There is still urgent need for a vaccine against schistosomiasis, especially in Schistosoma japonicum endemic areas where even a vaccine that will interrupt zoonotic transmission will be potentially effective as an intervention tool. We had developed a novel nanoparticle gene delivery system, which has proven efficacious in gene transfection to target immune cells with complementary adjuvant effect and high protective efficacy in several diseases. Here, we applied this nanoparticle system in combination with S. japonicum glutathione S-transferase (SjGST) DNA vaccine to show the immunogenicity and anti-fecundity effect of the nanoparticle coated vaccine formulation against murine schistosomiasis. The nanoparticle-coated DNA vaccine formulation induced desired immune responses. In comparison with the nanoparticle coated empty vector, it produced significantly increased antigen-specific humoral response, T-helper 1 polarized cytokine environment, higher proportion of IFN-γ producing CD4(+) T-cells and the concomitant decrease in IL-4 producing CD4(+) T-cells. Although there was no effect on worm burden, we recorded a marked reduction in tissue egg burden. There was up to 71.3% decrease in tissue egg burden and 55% reduction in the fecundity of female adult worms. Our data showed that SjGST DNA vaccine, delivered using the nanoparticle gene delivery system, produced anti-fecundity effect on female adult schistosomes as previously described by using conventional subunit vaccine with adjuvant, proving this DNA vaccine formulation as a promising candidate for anti-pathology and transmission blocking application., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Conflicting reports are found in the literature about the antischistosomal efficacy of Mirazid (MZ), which is a special formulation of myrrh obtained from the stem of the plant Commiphora molmol. This initiated the present study to assess this drug for the first time in experimental schistosomiasis japonicum. Mice were divided into four groups: infected untreated control (I); infected treated with MZ, 500 mg/kg (II); infected treated with MZ, 250 mg/kg (III); and infected treated with praziquantel (PZQ), 200 mg/kg (IV). The drugs were given 7 weeks post-infection for five successive days. All animals were killed 3 weeks posttreatment. Results showed no signs of antibilharzial activity of MZ. Total worms, total tissue egg load, egg developmental stages, and granuloma area were not affected by any of the MZ treatment regimens as compared to the infected untreated group (P > 0.05 for all variables). These results were in contrast to those obtained in PZQ-treated animals in which 82.82 % total worm reduction, 94.62 % egg reduction, and 86.35 % granuloma area reduction were observed. Also, it significantly increased the percentage of dead ova and decreased the percentage of mature ova with complete absence of immature ones in comparison with the control group (P < 0.01 for all variables). In conclusion, the results of the current study raise serious doubts about the antischistosomal activity of MZ.

Due to the importance of neutrophils and proinflammatory cytokines in schistosomal liver damage, we analyzed the mechanisms underlying neutrophil and proinflammatory responses in murine schistosomiasis japonica. We found that granulomatous inflammation around parasite eggs in the liver was greater in Schistosoma japonicum-infected IL-4-/- IL-13-/- (double-knockout [DKO]) mice than in infected wild-type (WT) mice at 6 weeks, but not at 8 weeks, postinfection, suggesting the importance of Th2 responses in these typical hepatic lesions. Infected DKO mice also showed increased neutrophil infiltration accompanying more severe pathology, as shown by the enhanced necrosis of hepatocytes. This was not likely due to a Th1/Th2 imbalance, because there was no detectable increase in gamma interferon (IFN-γ) production in these DKO mice. mRNA expression of interleukin-17A (IL-17A), proinflammatory cytokines, and the neutrophil chemoattractant CXCL2 in liver was higher in infected DKO mice than in WT mice. However, in IL-4-/- IL-13-/- IL-17A-/- (triple-knockout [TKO]) mice, the absence of IL-17A was associated with only marginal differences in schistosomal liver damage, suggesting that IL-17A is only partially responsible for neutrophil-driven hepatic damage. Furthermore, the expression of mRNAs encoding proinflammatory cytokines was not under the control of IL-17A in TKO mice. These findings indicate that IL-4 and IL-13 suppress excessive neutrophil recruitment, proinflammatory cytokine production, and hepatic damage during the acute stage of S. japonicum infection, suggesting that neutrophils and proinflammatory cytokines are mainly responsible for hepatocyte damage during acute murine schistosomiasis japonica. However, neutrophil induction and the production of proinflammatory cytokines were not due solely to IL-17A.

Background/aims: The mortality due to hepatocellular carcinoma (HCC) has ranged widely in various areas of Japan since 30 years ago and the incidence was particularly high in once Schistosoma japonicum (Sj)-endemic areas. Our aim was to estimate the spread time of hepatitis C virus (HCV) infection in the past with possible relevance to a higher incidence of HCC in once Sj-endemic than Sj-nonendemic areas., Methods: During 2001, 131 strains of HCV-1b were obtained from patients in three previously Sj-endemic areas, as well as Sj-nonendemic areas in Japan and a cross-sectional study was conducted on them with molecular evolutionary analyses., Results: A phylogenetic tree reconstructed on HCV-1b sequences in the NS5B region disclosed 2 independent clusters for Sj-positive and -negative groups with a high bootstrap value. The estimated effective number of HCV-infections indicated a transition from quiescence to rapid exponential growth in the 1920s among patients with schistosomiasis, which is 20 years earlier than that among patients without schistosomiasis., Conclusions: The estimated spread time in previously Sj-endemic areas in Japan coincides with injection treatment for Sj since 1921. A high incidence of HCC there would be attributed to a long duration of HCV infection since 1920s.

Vaccine development by the use of calpain of Schistosoma japonicum has been tried in our laboratory. We cloned cDNA encoding the heavy chain of S. japonicum calpain, and prepared recombinant molecule of a possible vaccine region of the heavy chain. When BALB/c mice were immunized with our recombinant calpain of S. japonicum with Freund's complete adjuvant, we observed significant reduction in worm burden (41.2% reduction, P<0.05), and also significant anti-fecundity effects. In this sense, calpain of S. japonicum seems to have infection control as well as anti-disease effects. Mechanisms of vaccine effects of calpain remain to be clarified, however, several effector mechanisms are suspected. In immunized mice, raised level of iNos expression was observed, while adhesion of peritoneal exudates cells were also observed in the presence of calpain-immunized sera, suggesting the possibilities of both cellular and humoral protective mechanisms. We examined tissue distribution of calpain in various developmental stages of S. japonicum. Strong signal was observed around excretory grand of cercariae, and they secreted calpain during their migratory movement tested in vitro. Together with the findings, calpain seems to induce larvicidal effects in the immunized mice. We observed time-course kinetics of antibody production against vaccine candidates in experimental S. japonicum infection in pigs. Although significant levels of antibody production were observed for paramyosin and GST, no significant antibody production was observed for calpain. This suggests that calpain is less immunogenic, and route of immunization and/or choice of adjuvant are important in future trials of calpain vaccine.

We summarized historical aspects of disease control activities targeting schistosomiasis japonica in Kofu basin, Yamanashi Prefecture, Japan. Kofu Basin was one of the biggest endemic foci of schistosomiasis japonica in Japan, and the last place where transmission of Schistosoma japonicum was confirmed in Japan. Because of the most severe endemic situations in Yamanashi, intensive control measures had been implemented by the central as well as the local government. The last human case in Japan was in 1977, which is just before praziquantel being available. Therefore, the main efforts were focused on snail control. Mass examination and mass chemotherapy were implemented, however, the compliance was not so good because of the severe side effects due to the available therapeutics, Stibnal. Along with socioeconomic development after World War II, big changes in land use, life style, and farming led drastic reduction in the disease prevalence in Kofu Basin in the 1960s. A large amount of budget was also used for disease control. Cementing water canals covered more than 95% of paddy fields in Kofu Basin, and this resulted in ecological changes. After elimination of schistosomiasis, environmental repair is the urgent subject in Kofu Basin. Our experiences in Yamanashi contain both good influences and also a lot of reflection. It is important to evaluate each activity in our history before we give intensive cooperation with countries where endemic foci is still present.

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Schistosoma japonicum soluble egg antigens (SEA) in un-concentrated urine was developed. The urine ELISA was applied to samples collected in a schistosomiasis-endemic village in China. The levels of anti-SEA antibodies detected in urine correlated well with those obtained with paired serum samples (r = 0.694, p<0.0001). Among 129 serum ELISA positives, 112 (86.8%) were positive by urine ELISA, while all 40 serum ELISA negatives from a non-endemic area were negative. The levels of anti-SEA in urine samples were stable up to 8 weeks of storage at 37 degrees C, with sodium azide as a preservative. Therefore, ELISA with urine samples can be used for the surveillance of schistosomiasis.

In our attempt to discover a potential cause for accumulation of cholesteryl ester transfer protein (CETP) deficiency in Eastern Asia, we studied the association of CETP deficiency with pathogenesis of Schistosoma japonicum, a life-threatening parasite peculiar to this region. The eggs of S. japonicum showed slow embryonation when cultured in CETP-deficient human plasma. Restoration of CETP to the deficient plasma rescued it, while inhibition of CETP in normal plasma did not cause slow embryonation of the cultured eggs. The egg embryonation was also retarded in the liver but not in the intestine of wild-type mice in comparison to the CETP-transgenic mice. The granulomatous lesion around the parasite eggs in the liver was less in the wild-type than in the CETP-transgenic mice. Thus, CETP deficiency may act against Schistosomiasis japonica by retarding egg embryonation, a potential cause of liver granulomatosis. It does not seem directly due to the lack of CETP activity in plasma but to abnormal lipoprotein generated by chronic CETP deficiency., (Copyright 2001 Academic Press.)

Mice infected with Schistosoma japonicum were resistant to the intestinal nematode, Strongyloides venezuelensis. The numbers of adult S. venezuelensis recovered from mice were significantly decreased when infections were given from 6 weeks after S. japonicum infection. Larval recovery from the lungs showed that significant numbers of subcutaneously inoculated S. venezuelensis larvae were eliminated by 3 days in S. japonicum-infected mice (P < 0.0001), while histology revealed that this was associated with massive eosinophilic infiltration in the lungs. In addition, adult S. venezuelensis worms implanted in the duodenum of S. japonicum-infected mice could not establish in the intestine. This failure was associated with mucosal mastocytosis. Activation of eosinophils and intestinal mast cells was correlated with elevated expression of mRNA for interleukin (IL)-3, IL-4, and IL-5 in S. japonicum-infected mice. Sera from S. japonicum-infected mice recognized S. venezuelensis larva antigens as strongly as those from S. venezuelensis-infected mice, although transfer of sera from S. japonicum-infected mice to normal recipient mice did not protect them from S. venezuelensis challenge infection. It was concluded that the mechanisms for larval killing and adult worm expulsion of S. venezuelensis in S. japonicum-infected mice were identical to those seen in infections with S. venezuelensis only.

Objective: To determine the effect of Schistosoma japonicum infection on the testoterone level in the sera from male C57BL/6 mice., Methods: Radioimmunoassay was used to examine testosterone levels in sera of 9 male mice experimentally infected with Schistosoma japonicum., Results: The serum testosterone levels reduced significantly in all experimentally infected animals 45 days after infection, as compared with the uninfected controls., Conclusion: Infection with Schistosoma japonicum decreases testosterone levels in the mouse host.

Mutations in p53 tumor suppressor gene were examined in 44 Chinese patients with rectal cancer, including 22 cases with advanced schistosomiasis japonica and 22 cases without schistosomiasis. In schistosomal rectal cancer (SRC), 13 mutations were found in 10 cases, which included 11 base-pair substitutions and two deletions. Of 11 base substitutions, nine were transitions and two were transversions and seven of them were located at CpG dinucleotides. In non-schistosomal rectal cancer (NSRC), 13 mutations were found in nine cases, all of which were base-pair substitutions. Of 13 substitutions, 10 were transitions and three were transversions and three of them were located at CpG dinucleotides. The proportion of base-pair substitutions at CpG dinucleotides was higher in SRC patients than in NSRC patients, although this was not statistically significant (P = 0.054). Point mutation was frequent at codon 248 in SRC. A higher frequency of arginine missense mutations was observed in SRC than in NSRC. These observations suggest that the mutations in SRC are the result of genotoxic agents produced endogenously through the course of schistosomiasis japonica.

This article contains a review of current knowledge on the association of parasite infections and cancer formation, especially that of Schistosoma japonicum (Trematoda) in man and experimental animals. The association of S. haematobium infection and bladder cancer is well known and documented. However, S. japonicum infection has also been reported to be associated with cancer, in this case hepatocellular carcinoma and/or colorectal cancer. Pathological records and analyses have shown a correlation between this infection and cancer, and pathohistological descriptions have been numerous, together with clinical case reports. Epidemiological analyses have been conducted in China and Japan and support a role of S. japonicum infection as one of the risk factors in cancer formation, along with others, such as hepatitis virus infection and alcoholic intake. Experimental results have also shown that cancer appears early and in larger numbers in experimentally infected animals given a known carcinogen. In spite of these positive end-point associations, the mechanism of schistosome-mediated enhancement of carcinogenesis is obscure. A suggestive observation is that in S. japonicum-infected mice carcinogen-metabolizing hepatic activity including P-450 was decreased so that an administered carcinogen persisted for a longer period than in uninfected mice. Further studies, both epidemiological and experimental, are needed to firmly establish the relationship between schistosome infection and cancer.

Immunogenetic factors were studied in 60 patients with schistosomiasis japonica in the Philippines, of whom 15 were characterized by marked hepatosplenic lesions and 45 characterized by cerebral symptoms. Immune responsiveness of the patients to schistosomal antigen was measured by T cell proliferation in vitro, and their HLA-A and -B specificities were typed. All but one hepatosplenic patients showed strong immune responsiveness to the schistosomal antigen, whereas both low and high responders were observed in the cerebral patients. A significant association between HLA-B40 and high responders to the schistosomal antigen was observed (P = 0.0458), and this HLA specificity was increased in frequency in the hepatosplenic patients. HLA-B16 was not observed in the hepatosplenic patients, but was common in the cerebral patients (26.5%) (P = 0.0255), and this HLA specificity was commoner in the low responders than in the high responders. These observations suggest that an HLA-linked gene governs the clinical manifestations of human schistosomiasis japonica by controlling immune responsiveness of the infected hosts to the schistosomal antigen.

To characterize the mechanisms of induction and regulation of the cell population involved in granuloma formation around eggs of Schistosoma japonicum, we utilized a simple method of in vitro experiments. Lyt1+2-T cells were essential for in vitro responses to the intact S. japonicum eggs, which were assumed to be comparable to in vivo granulomatous responses. T-cell responses seemed to be macrophage-dependent, and responding T cells produced IL-2-like activities in the culture supernatant. Sera taken from chronically infected mice acted as regulatory factors to these T-cell responses as was the case in in vivo granuloma formation. The method used here was simple and highly informative for the studies on pathogenesis of schistosomiasis japonica.

We tested effects of praziquantel, an antischistosomal compound, on clinical and immunological parameters of chronic schistosomiasis japonica. Two Japanese patients, who had high antibody titers to Schistosoma japonicum antigens but no fecal schistosome eggs or no or mild symptoms complained, were treated with praziquantel. Within two years after treatment, anti-schistosome antibodies in sera from the patients became negative in enzyme-linked immunosorbent assay. There was no significant alteration in cellular immunity to the parasite. Although S. japonicum infection is believed to have been eradicated in Japan, our present results seem to suggest the possibility that a few Japanese individuals, who have high anti-schistosome antibody, still harbor live parasites.

Antigens that produce an antibody response in some members of a species may fail to do so in others. The response to an antigen is controlled by a gene termed the immune response (Ir) gene, which is transmitted as a single dominant trait. We have provided evidence for similar immune suppression (Is) genes which control non-responsiveness through the antigen specific suppressor T cell. The non-responsiveness is also dominantly inherited and the Is genes are linked to the histocompatibility (HLA) antigen system. Here we report that the HLA-DR2 molecule from a non-responder haplotype (HLA-Dw12-DR2-DQwl) is required for the proliferative T cell response to schistosoma japonicum (Sj) antigen, as a restriction element, indicating that the HLA-DR2 is the product of the Ir gene, and that the HLA-DQwl molecule of the non-responder haplotype is important in the antigen-specific suppression of the response to this antigen, suggesting that it is the product of the Is gene. We therefore conclude that the HLA-DR and DQ molecules, which are controlled by the distinct genes in the MHC multigene family, regulate immune response and immune suppression and that the gene for HLA-DQ is epistatic to that for HLA-DR in controlling the immune response to schistosomal antigen in humans.

T cell lines (TCLs) specific for Schistosoma japonicum egg antigen were established from a patient with chronic schistosomiasis japonica to investigate the regulatory mechanism of S.japonicum egg antigen-driven T cell responses in man. All five TCLs tested were CD2+, CD4+, CD8-, and were strongly proliferative only to S. japonicum egg antigen in the absence of exogenous IL-2. All but one TCL produced IL-2-like lymphokines in vitro, indicating their helper T cell functions. One TCL, SjE-3, failed to produce IL-2-like lymphokines. Moreover, this TCL suppressed the specific proliferation of autologous peripheral blood lymphocytes to S. japonicum egg antigen. This TCL produced a soluble suppressor factor(s). These functional diversities among established TCLs were also confirmed by cloned T cells. Our observations might suggest that the regulatory system through helper and suppressor T-T interactions somehow involved in T cell responses to the egg antigen in human chronic schistosomiasis japonica.