Schistosoma malayensis n. sp.: a Schistosoma japonicum-complex schistosome from Peninsular Malaysia

Schistosoma malayensis n. sp., a member of the Schistosoma japonicum complex is described from Rattus muelleri in Peninsular Malaysia and 2 strains are characterized. The only morphological differences noted among adults from natural hosts were that S. malayensis are in general smaller than S. mekongi and S. japonicum. But these differences may be the result of host-induced variations and therefore are of little taxonomic value. To minimize the effects of host-induced variations, adult worms recovered from laboratory mice with similar worm burdens at 50-56 days postinfection were compared. These comparisons revealed only minor morphometric differences among these 3 species. Schistosoma malayensis eggs from naturally and experimentally infected hosts are most similar to those of S. mekongi, with eggs of both species being, in general, smaller than those of S. japonicum. The egg index for S. malayensis is usually higher than for S. japonicum and lower than for S. mekongi. Differences were noted in the developmental rates in mice for 2 isolates of S. malayensis, S. mekongi, and S. japonicum (Philippine strain), but relatively large differences observed between isolates of S. malayensis indicate that, in this case, the developmental rate is not a useful taxonomic character. Schistosoma malayensis is erected principally on the basis of differences, reported elsewhere, in the life histories and in the electrophoretic migration patterns of isoenzymes of adult worms as compared to S. mekongi and S. japonicum. These comparisons indicate that S. malayensis is more closely related to S. mekongi than to S. japonicum.     

Schistosoma mekongi: a prominent neutrophil chemotactic activity of egg antigen with reference to that of Schistosoma japonicum

Schistosoma mekongi causes granulomatous lesions around eggs deposited in the liver with neutrophil-rich inflammatory reactions in the early stage of the egg laying. To define the aspects of the typical pathogenesis of S. mekongi infection, we determined the difference between soluble egg antigen (SEA) from S. mekongi and S. japonicum with a focus on chemotactic factors for neutrophils or eosinophils. Mean volume and protein amount of S. mekongi eggs was 71 and 58% of those of Schistosoma japonicum eggs, respectively. Neutrophil chemotactic activity of S. mekongi SEA was about two times higher than that of S. japonicum. In contrast, eosinophil chemotactic activity of S. mekongi SEA was about half of that of S. japonicum SEA. Molecular analysis revealed that S. mekongi SEA contains higher molecular-weight components with a lower level of glycosylation, and this is likely to be related to the intense neutrophil chemotactic activity in comparison with S. japonicum SEA. The prominent chemotactic reactivity for neutrophils is likely to be involved in the typical pathogenesis of mekongi schistosomiasis.     

The pig as a host for Schistosoma mekongi in Laos.

A survey of helminths in domestic pigs was conducted in Khong District, Laos, to elucidate if these domestic animals could act as definitive hosts for Schistosoma mekongi and to obtain a general overview of their helminthological infection status. Fecal samples were collected from 98 pigs. Twelve pigs (12.2%) were found to excrete S. mekongi eggs. Infection was confirmed by detection of S. mekongi eggs in tissues of liver, rectum, and cecum of 2 pigs. A total of 75.8% of the pigs was infected with 1 or more helminth species. This study showed that pigs may act as a definitive host for S. mekongi.     

Development of a real-time PCR assay with fluorophore-labelled hybridization probes for detection of Schistosoma mekongi in infected snails and rat feces

SUMMARY Schistosoma mekongi, a blood-dwelling fluke, is a water-borne parasite that is found in communities along the lower Mekong River basin, i.e. Cambodia and Lao People's Democratic Republic. This study developed a real-time PCR assay combined with melting-curve analysis to detect S. mekongi in laboratory setting conditions, in experimentally infected snails, and in fecal samples of infected rats. The procedure is based on melting-curve analysis of a hybrid between an amplicon from S. mekongi mitochondrion sequence, the 260 bp sequence specific to S. mekongi, and specific fluorophore-labelled probes. This method could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails, a single cercaria in filtered paper, and 2 eggs inoculated in 100 mg of non-infected rat feces. All S. mekongi-infected snails and fecal samples from infected rats were positive. Non-infected snails, non-infected rat feces, and genomic DNA of other parasites were negative. The method gave high sensitivity and specificity, and could be applied as a fast and reliable tool for cercarial location in water environments in endemic areas and for epidemiological studies and eradication programmes for intermediate hosts.     

Schistosoma japonicum infection in pregnant mice.

Ten 1-week and ten 2-weeks pregnant female NMRI mice were experimentally exposed to 70 Schistosoma japonicum cercariae. Ten littermice from each group were examined for worms by perfusion 4, 6 and 8 weeks post infection. Although the mothers (n = 15) were found infected with 15.5 +/- 13.4 worms at perfusion 6 and 7 weeks post infection, no worms were found in any of the examined littermice, as well as no detection of faecal or tissue eggs. Litter sizes did not differ from control groups and all littermice were healthy. The present study therefore suggests that congenital infection with S. japonicum does not occur in percutaneously infected mice and that infection of the mother during pregnancy does not seem to affect the offspring.     

Schistosoma japonicum: the pathology of experimental infection

The pathology of experimental schistosomiasis japonica is reviewed and compared with the pathology of schistosomiasis japonica in man and to some aspects of schistosomiasis mansoni and schistosomiasis haematobia in experimental animals. The induction of granulomas around Schistosoma japonicum eggs depends upon cell mediated immunity, as do the reactions to Schistosoma mansoni and Schistosoma haematobium eggs. However, the modulation of the reaction to S. japonicum eggs can be greatly influenced by antibody, while antibody has no effect on the granulomas around S. mansoni eggs. Adult worm pairs of S. japonicum tend to cluster in the mesenteric venules, and most eggs are laid in a few sites. This leads to large, focal intestinal lesions similar to the discrete lesions produced by S. haematobium in the intestine and urinary tract but in contrast to the widespread, diffuse lesions produced by S. mansoni. Comparison with S. japonicum infection in humans is limited chiefly by our scant knowledge of the pathology produced by S. japonicum in infected persons. Most such comparisons are, in any case, limited by the marked differences in the reactions of various experimental host species to the infection and by differences in the reaction of a given host species to different strains of the parasite.     

Adverse effects of praziquantel treatment of Schistosoma japonicum infection: involvement of host anaphylactic reactions induced by parasite antigen release

The present study using a murine model heavily infected with Schistosoma japonicum aimed to elucidate the pathogenesis of adverse effects of praziquantel treatment of schistosome-infected subjects. Inbred BALB/c mice were infected with S. japonicum (Yamanashi strain) before being treated with a single dose of praziquantel at 4 or 8 weeks p.i. All the mice treated at 8 weeks p.i. exhibited signs typical of systemic anaphylaxis until half of them died shortly after praziquantel administration. At autopsy, these mice exhibited remarkable intestinal alterations characterised by increased mucosal permeability, mucosal oedema and petechial haemorrhage, which are changes typical of immediate intestinal anaphylaxis. In these mice treated at 8 weeks p.i., degranulation of intestinal mast cells was frequently observed, which was particularly remarkable around S. japonicum eggs hatched as an effect of praziquantel. Furthermore, the plasma histamine concentration just after praziquantel treatment was much higher in mice at 8 weeks p.i. than that in uninfected mice or in S. japonicum-infected mice without drug treatment. In contrast, none of these intestinal changes was observed in untreated or uninfected control mice, or in mice administered praziquantel at 4 weeks p.i., in which worm pairs had just reached sexual maturation and begun egg-laying. The finding by ELISA that serum IgM and IgA levels specific to S. japonicum eggs decreased immediately after praziquantel treatment, together with the results of immunohistochemistry, revealed the sudden release of parasite antigens from the eggs hatched by praziquantel treatment. The results of this study demonstrate that adverse effects of praziquantel treatment of schistosomiasis characterised by abdominal signs depend on anaphylactic reactions due to parasite antigens, especially antigens from eggs hatched as an effect of praziquantel.     

Schistosoma mansoni, S. haematobium, and S. japonicum: identification of genus- and species-specific antigenic egg glycoproteins

Immunoreactive egg glycoproteins of Schistosoma mansoni, S. haematobium, and S. japonicum which are genus- and species-specific, or react with sera of patients infected with other parasites, have been identified. Egg proteins were labeled with Iodine-125, and the concanavalin A-binding glycoproteins were immunoprecipitated with sera of patients infected with one of four species of Schistosoma or Trichinella spiralis, Taenia solium, Echinococcus granulosus, Entamoeba histolytica, or Wuchereria bancrofti. These immunoprecipitates were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Despite the strikingly different patterns of glycoproteins of the African species, the antibody immune responses of patients infected with S. mansoni and S. haematobium were found to be so similar that differentiation could not be established. In contrast, sera of patients infected with S. japonicum, S. mekongi, or parasites not of the genus Schistosoma, immunoprecipitated fewer of the major S. mansoni or S. haematobium glycoproteins. Likewise, antibody immune responses of patients infected with the Oriental schistosomes (S. japonicum and S. mekongi) could not be differentiated. Only a few quantitative differences were noted between our S. mansoni egg glycoprotein extract and a standardized soluble egg antigen extract. This study provides an explanation for the extensive cross-reactivity observed in diagnostic assays which utilize various fractions of schistosomal egg extracts as the antigen.     

Congenital infection with Schistosoma japonicum but not with Schistosoma bovis in sheep

The present study investigated whether Schistosoma japonicum or Schistosoma bois could establish prenatally in lambs. Three ewes were exposed to S. japonicum by intramuscular injection of cercariae, and 3 ewes were exposed to S. bovis cercariae using the leg-emerging technique approximately 2 mo before delivery, and 1 age-matched pregnant ewe served as an uninfected control. The study lasted 18-20 wk after infection, which was 8-9 wk after delivery. All 6 exposed ewes became infected with either S. bovis or S. japonicum. Eight lambs were borne by the 7 ewes, of which 1 (S. bovis exposed) was dead and 1 (S. japonicum exposed) died at delivery. Of the 3 S. japonicum-exposed lambs, 2 were found infected. Four lambs born of S. bovis-exposed ewes were negative. Despite having no worms, these 4 S. bovis-exposed lambs as well as the 1 negative S. japonicum-exposed lamb had, in contrast to the nonexposed control lamb, few, but distinct, liver granulomas dominated by eosinophils and giant cells with large central necrotic areas but with no remnants of eggs or worms. Hence, congenital infection was demonstrated in S. japonicum-infected lambs, but not in S. bovis-infected ones.     

Efficacy of administration of praziquantel on 2 days 2 weeks apart against Schistosoma japonicum eggs in mice

We compared a group of mice given multiple oral doses of praziquantel (PZQ) on 1 day 7 weeks after infection with Schistosoma japonicum and another group given multiple doses of PZQ over 2 days (7 and 9 weeks after infection) by examining the number of schistosome eggs and oograms in their tissues and feces. In the 1-day-protocol group (4 x 100 mg/kg x 1 day), calcified dead eggs or shells accounted for 77.4 and 70.1% of the total EPG (the total number of immature eggs, mature eggs, and calcified eggs and shells per 1 g tissue) in the liver and small intestine, respectively, 11 weeks after infection. Shells accounted for nearly half of the total EPG (54.3% liver and 46.6% small intestine). Mature eggs accounted for 8.4% (liver) and 5.1% (small intestine) of the total EPG. Only shells were found in the feces. In the 2-day-protocol group (4 x 100 mg/kg x 2 days, 2 weeks apart), dead eggs accounted for 87.2 and 89.5% of the total EPG in the liver and small intestine, respectively, 11 weeks after infection. Shells accounted for 62.5% (liver) and 75.8% (small intestine) of the total EPG. In the 2-day group, mature eggs accounted for 0.9% (liver) and 0.6% (small intestine) of the total EPG. In liver and small intestine, the EPG of immature and mature eggs in the 2-day group was significantly smaller than in the 1-day group. Especially, the tendency was clear in the case of the EPG of mature eggs. There were no schistosome eggs in the feces of the 2-day group. We found that administration of PZQ over 2 days separated by a 2-week interval was effective against S. japonicum.     

Schistosoma japonicum and Paragonimus ohirai: antagonism between S. japonicum and P. ohirai in Oncomelania nosophora

Antagonistic interactions between Schistosoma japonicum and Paragonimus ohirai were examined in the snail host, Oncomelania nosophora. When P. ohirai-infected snails were exposed to S. japonicum miracidia at intervals of 4 to 18 weeks post-first exposure, only a few snails (0-7%) were found to be superinfected with S. japonicum sporocysts. Sporocysts were fewer in number than those of single infected controls. Mature S. japonicum cercariae were not observed. Furthermore, when the snails were examined at intervals of 14 to 18 weeks post-second exposure, neither sporocysts nor cercariae of S. japonicum were found. On the other hand, when the snails were exposed to miracidia of S. japonicum and P. ohirai simultaneously, they were easily infected with both parasites. At 26 weeks after simultaneous exposure, however, the infection rate of S. japonicum was significantly lower than that of controls. In contrast, when S. japonicum-infected snails were exposed to P. ohirai miracidia, they were superinfected with P. ohirai, although the infection rate was somewhat lower than that of controls. These results indicate the existence of antagonism between S. japonicum and P. ohirai in O. nosophora. Furthermore, P. ohirai was dominant over S. japonicum in the antagonistic interactions in this snail host.     

Molecular phylogenetic position of Schistosoma sinensium in the genus Schistosoma

The status of Schistosoma sinensium (samples from Thailand and from Sichuan, China) relative to other species of the genus Schistosoma was investigated using DNA sequences from the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene (partial) and the nuclear ribosomal DNA second internal transcribed spacer 2 (ITS2). Trees inferred from these sequences place S. sinensium as sister to the S. japonicum group and suggest a basal position in the clade utilizing snails of the family Pomatiopsidae. The sequence differences between specimens of S. sinensium from China and Thailand are at least as great as between S. malayensis and S. mekongi. Schistosoma sinensium is probably best regarded as a species complex.     

东方人类血吸虫及其螺蛳宿主交互感染的研究

以我国安徽及菲律宾莱特岛的日本血吸虫、湄公血吸虫、马来西亚类似日本血吸虫以及中华血吸虫交互感染其螺蛳宿主。结果表明日本血吸虫及钉螺与湄公血吸虫、马来西亚类似日本血吸虫及其螺蛳宿主亲缘关系较疏远。而湄公血吸虫与马来西亚类似日本血吸虫及其螺蛳宿主有较近的亲缘关系。交互感染结果为马来西亚类似日本血吸虫及钙河罗氏螺的分类问题提供了新的依据。     

Dynamic analysis of T-lymphocyte function in relation to hepatopathologic changes and effect of interleukin-12 treatment in mice infected with Schistosoma japonicum

We analyzed the dynamics of splenic T-lymphocyte function in relation to hepatopathologic changes in C3H/Hc mice, experimentally infected with Schistosoma japonicum. Vigorous granuloma formation was observed at 7 wk postinfection. At 10 wk postinfection, granuloma formation entered into the down-modulation stage, as represented by the diminished granuloma size. The Th2 response was activated when eggs appeared in the liver, whereas Th1 responses were depressed and the proliferation of T lymphocytes was decreased. The level of IgG antibodies to the worm and egg antigens rose continually after infection. Interleukin-12 treatment of infected mice inhibited Th2 responses and T-cell proliferation, decreased granuloma formation and fibrosis, but had no effect on the fecundity of the worms. These data suggest that egg deposition is the major factor driving Th2 responses, depressing Th1 cytokine expression as well as T-cell proliferation in S. japonicum-infected mice.     

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.     

Immune changes of Schistosoma japonicum infections in various rodent disease models

Rodent models for Schistosoma japonicum infections have demonstrated that these animals possess a degree of resistance to schistosome infections that may be both T and B lymphocyte-mediated. However, their exact role is not well-defined and other immune mechanisms are likely to also play a role in protecting against infection. Immunosuppressed and immunocompetent reed voles (Microtus fortis, Mf), rats and mice (n=24/group) were infected with S. japonicum, and animals were sacrificed 42 days later under anesthesia. Neither worms nor eggs were observed in infected immunosuppressed Mf or rats, with the exception of one rat that presented with few eggs. In immunosuppressed mice, changes in the number and size of the worms were not significantly different compared to immunocompetent mice, but worm fecundity was affected. The size and number of granulomas in immunosuppressed animals was also reduced. Analysis of serum antibodies specific to schistosome adult worm antigen at 3 weeks post-infection demonstrated that the levels of antibodies in the sera of rats were significantly higher than in Mf and mice. In addition, Mf serum levels of IL-4 and IL-12 were significantly higher than levels observed in rats and mice. Antibodies and cytokines in the sera of Mf peaked 3 weeks post-infection and then began to decrease, while antibody responses in rats and mice increased gradually between weeks 3-7 post-infection. It is possible that T and B cells have a dual role in both mediating protection and exacerbating disease outcomes.     

Schistosoma japonicum: immunological characterization and detection of circulating polysaccharide antigens from adult worms

The antigenic constituents of a trichloroacetic acid (TCA)-soluble fraction of adult Schistosoma japonicum were studied with immunoelectrophoresis, and compared with those of Schistosoma mansoni. Eight TCA-soluble antigens of S. japonicum were demonstrated, five of which showed immunological identity with S. mansoni antigens. Of the eight antigens, five antigens with anodic motility were found as circulating antigens in S. japonicum-infected hamster and rabbit sera; the major circulating antigen was the circulating anodic antigen (CAA). Two other antigens, with cathodic motility, including the circulating cathodic antigen (CCA), were demonstrable as circulating antigens in S. mansoni infections, but not in S. japonicum infections. Most of the circulating antigens were shown to be gut-associated. Only one antigen, line 2, which was not demonstrable as circulating antigen and which was present in the parenchyma of the worms, was found to be specific for S. japonicum. Using an ELISA for the detection of CAA in the sera of S. japonicum-infected rabbits, a lower detection level of 100 ng CAA/ml serum was achieved. Moreover, at 7-8 weeks after infection, a direct relationship between worm burden and CAA level was demonstrated.     

Reduced levels of mutagen processing potential in the Schistosoma japonicum-infected mouse liver

Epidemiological studies have shown the presence of a positive correlation between the infection of Schistosoma japonicum and colorectal and/or liver cancers in the humans. To explore the mechanism underlying this correlation, we have investigated the mutagen-activating potentials of the liver homogenate fraction (S9) from Schistosoma japonicum infected mice and those from control mice, by use of the Ames test with 2-acetylaminofluorene, aflatoxin B1 and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) as test mutagens. Liver S9 prepared from the infected group at the 15th week after the infection showed a potential significantly lower than that from the control group. The hepatic cytochrome P-450 concentration in the infected mice was persistently low, about a half of that in the uninfected mice, during the period of 6-18 weeks after the infection. Thus, in mice bearing chronic schistosomiasis, mutagen-processing potentials are decreased.     

Prevention of Schistosoma japonicum infection in mice with long-acting praziquantel implants

This work reports the prevention outcomes of a praziquantel (PZQ) implant against the infection of Schistosoma japonicum in mice. The PZQ implant produced stable plasma PZQ concentrations in a range of 100-1300 ng/mL for a period of 70 days, by releasing PZQ in subcutaneous tissues in a sustained manner. To assess the prevention effects, the mice were infected at varying times after implantation. All the mice were sacrificed at 6 weeks after infection for worm and egg recovery and counting, worm morphological examination, determination of egg-hatching rates, and analysis of hepatic histology. The infection was successfully prevented for mice with early infection times (within 2-3 weeks), as nearly no worms, paired worms, eggs, or miracidia were recovered. However, in mice with late infection times (after 3 weeks), the prevention effects were diminished due to the decreased plasma PZQ concentrations at late times. Interestingly, the implants showed robust prevention effects on repeated infection at 1 and 3 weeks. In the infection-prevented mouse livers, no granuloma formation or granulomatous inflammation was observed. The results demonstrated that by blocking the development of infecting miracidia and by deactivating the eggs, the PZQ implants encouragingly prevented the S. japonicum infection and avoided liver damage.     

Lack of effect of unmated schistosomes on the fecundity of mated worm pairs

An imbalance between the numbers of male and female worms had no significant effect upon the fecundity of Schistosoma mansoni in rhesus monkeys or upon the number of S. mansoni eggs in the tissues of infected persons or mice. The number of S. japonicum eggs in the tissues of infected rabbits was similarly unaffected by disproportion between worm genders.