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The cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, S. proliferum’s phylogeny and life cycle remain enigmatic. To investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the mechanisms underlying pathogenicity, we sequenced the entire genomes of S. proliferum and a closely related non–life-threatening tapeworm Spirometra erinaceieuropaei. Additionally, we performed larvae transcriptome analyses of S. proliferum plerocercoid to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum has experienced a clearly distinct evolutionary history from S. erinaceieuropaei. Moreover, we found that nonordinal extracellular matrix coordination allows asexual reproduction in the host, and loss of sexual maturity in S. proliferum are responsible for its fatal pathogenicity to humans. Our high-quality reference genome sequences should be valuable for future studies of pseudophyllidean tapeworm biology and parasitism.

The cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, S. proliferum’s phylogeny and life cycle remain enigmatic. To investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the mechanisms underlying pathogenicity, we sequenced the entire genomes of S. proliferum and a closely related non–life-threatening tapeworm Spirometra erinaceieuropaei. Additionally, we performed larvae transcriptome analyses of S. proliferum plerocercoid to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum has experienced a clearly distinct evolutionary history from S. erinaceieuropaei. Moreover, we found that nonordinal extracellular matrix coordination allows asexual reproduction in the host, and loss of sexual maturity in S. proliferum are responsible for its fatal pathogenicity to humans. Our high-quality reference genome sequences should be valuable for future studies of pseudophyllidean tapeworm biology and parasitism., Kikuchi et al. sequence the genome of the fatal tapeworm Sparganum proliferum and a closely related non–life-threatening tapeworm Spirometra erinaceieuropaei, and describe its genomic features suggesting the natural history and molecular mechanisms underlying pathogenicity. Their findings indicate that nonordinal extracellular matrix coordination is important for its asexual reproduction, and suggest that loss of sexual maturity contributes to the fatal pathogenicity of S. proliferum to humans.

BackgroundThe cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, the S. proliferum phylogeny and lifecycle are still an enigma.ResultsTo investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the underlying mechanisms of pathogenicity, we sequenced the entire S. proliferum genome. Additionally, S. proliferum plerocercoid larvae transcriptome analyses were performed to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum genetic sequence is distinct from that of the closely related Spirometra erinaceieuropaei. Moreover, nonordinal extracellular matrix coordination allows for asexual reproduction in the host and loss of sexual maturity in S. proliferum is related to its fatal pathogenicity in humans.ConclusionsThe high-quality reference genome sequences generated should prove valuable for future studies of pseudophyllidean tapeworm biology and parasitism.

We examined whether antibody isotype responses to paramyosin (PM), a vaccine candidate for schistosomiasis, are associated with age-dependent resistance and pathology in liver fibrosis using human sera collected from 139 individuals infected with Schistosoma japonicum in Leyte, The Philippines. We report that IgA and IgG3 responses to PM showed a positive correlation with age and that the epitopes responsible were localized predominantly within the N-terminal half of PM. In addition, the IgG3 response to PM was associated with serum level of procollagen-III-peptide (P-III-P), an indicator of progression of liver fibrosis. These results imply that IgG3 against PM may not only provoke age-dependent resistance to S. japonicum infection but also enhance liver fibrosis. In contrast, levels of IgE to PM and to multiple PM fragments showed a negative correlation with P-III-P level. Thus, in contrast to IgG3, increases in PM-specific IgE may contribute to suppression of liver pathogenesis in schistosomiasis.

Background The 29 kDa Schistosoma haematobium species-specific antigen (ShSSA) is of remarkable interest in the diagnosis of urinary schistosomiasis although it had not been fully characterized. Method To determine the biological importance of ShSSA in S. haematobium and pathogenesis of the disease, we immunolocalized ShSSA in schistosome eggshells, miracidia and adult worm sections using indirect fluorescent antibody test (IFAT). Results ShSSA was strongly immunolocalized in the schistosome eggshells, selective regions of the miracidia body and walls of internal organs such as oviduct, ovary, vitelline duct and gut of the adult worm. Conclusion The strong immunolocalization of ShSSA in schistosome eggshells and adult worm internal organs suggests that the antigens involved in the pathogenesis of urinary schistosomiasis could have originated from the eggs and adult worms of the parasite. The findings also indicate that ShSSA may play a mechanical protective role in the survival of the parasite.

The Asian Centre of International Parasite Control (ACIPAC) was established in Bangkok in March 2000 as a JICA technical cooperation project in collaboration with the Mahidol University and the Ministry of Public Health, Thailand. Since then, ACIPAC carried out its activities with emphasis on the establishment of the concept of school health-based parasite control in the Great Mekong sub-region countries. In addition to contributing human resource development through its training courses on school-based malaria and STH control for programme managers and also through small-scale pilot projects implemented in partner countries, ACIPAC proposed that schoolchildren should be considered as health partners rather than simple recipients of health services including deworming programmes.

Summary We tested a rapid visually read monoclonal antibody (MoAb) based dipstick assay for specific diagnosis of urinary schistosomiasis against microscopy and the use of haematuria and proteinuria in a schistosomiasis haematobia endemic area in the Central Region of Ghana. The study group consisted of 141 school children (83 males, 58 females) aged 8-19 years. A total of 129 of 141 (91.5%) submitted stool samples, and 7.8% had Schistosoma mansoni, 55% had hookworms and 6.2% had tapeworms. The presence of S. mansoni and intestinal parasites did not appear to influence the results of the MoAb-dipstick assay. The urinary schistosomiasis prevalence by MoAb-dipstick (78%) was higher (P < 0.05) than the estimate by microscopy (60.3%), microhaematuria (27%) and proteinuria (30.5%). The MoAb-dipstick correctly identified 98.8% of microscopically confirmed cases and missed one (1.3%). The dipstick was also positive for 26 of 56 (46.4%) egg-negative individuals, thereby giving a sensitivity of 98.8% and a specificity of 53.6%. On the other hand, microhaematuria and proteinuria were 38.8% and 30.6% sensitive, and 91.1% and 69.6% specific, respectively. Microhaematuria and proteinuria were less sensitive (P < 0.05) than both microscopy and MoAb-dipstick.

We used a rapid, visually read, field applicable monoclonal antibody (MoAb)-dipstick assay for specific diagnosis of urinary schistosomiasis together with microscopy to determine the prevalence of infant schistosomiasis in a community in the Awutu-Efutu Senya District in the Central Region of Ghana. The study group consisted of 97 infants (51 males and 46 females) aged 2 months to 5 years. A total of 75 of 97 (77.3%) subjects submitted stool samples; none had Schistosoma mansoni. Three individuals (3.1%) had hookworms but there were no other intestinal helminths. The urinary schistosomiasis prevalence by MoAb-dipstick (30%) was higher (P < 0.05) than that estimated by microscopy (11.2%). However, three of nine (33.3%) microscopically confirmed cases tested MoAb-dipstick positive after pre-treatment of the urine specimen with heat. The youngest infant to be found infected with S. haematobium microscopically was 4 months old. Fifteen of 71 S. haematobium egg negative individuals tested dipstick positive, giving a dipstick specificity of 78.9% as compared with microscopy as gold standard test. The relative sensitivity of the dipstick was 100%.

Schistosoma haematobium infection could be associated with morbidity. Generally, the cost of schistosomiasis control is high and it becomes a burden for governments or non-governmental organisations to repeat control programs so as to reduce morbidity. There is therefore, the need to optimise the available meagre resources for its control. From 1993 to 1997 the Noguchi Memorial Institute for Medical Research of the University of Ghana carried out a schistosomiasis control program in southern Ghana. Using the generated data, an attempt is made to determine the timing of the second praziquantel treatment and the period needed after the second chemotherapy to have egg counts reduced to low levels in southern Ghana. It was revealed that the second praziquantel treatment in areas 1, 2, and 3 should be administered latest at 13.8, 11.8 and 13.2 months, respectively after the first one. Most importantly, it takes 24.4 months to bring egg counts to zero in area 3 while in area 1, it takes about 29 months after the second praziquantel treatment. Egg counts were not reduced to zero in area 2 after the second chemotherapy. At least passive health education and continuous safe water supply should support the chemotherapy in addition to weed removal at the water contact sites.

A comparative study was carried out on cytokine and chemokine responses in a cerebral malaria (CM)-susceptible or -resistant strain of mice (C57BL/6 or BALB/c respectively) in Plasmodium berghei ANKA infection. C57BL/6 mice died by 10 days after infection when parasitemia was approximately 15-20% with cerebral symptoms, while BALB/c mice survived until week 3 after infection. Although both strains showed T(h)1-skewed responses on day 4 after infection, significantly higher levels of IFN-gamma, tumor necrosis factor (TNF)-alpha and NO were observed during the course of the infection in BALB/c, suggesting that T(h)1 responses are involved in the resistance. Interestingly, in the brain, both strains expressed IFN-inducible protein of 10 kDa (IP-10) and monocyte chemotactic protein (MCP)-1 genes as early as at 24 h post-infection, whereas some differences were observed between both strains thereafter, i.e. enhanced expression of RANTES in C57BL/6, and of IFN-gamma and TNF-alpha in BALB/c respectively. Moreover, the expression of IP-10 and MCP-1 genes in KT-5, an astrocyte cell line, was induced in vitro upon stimulation with a crude antigen of malaria parasites. These results suggest that the direct involvement of brain parenchymal cells takes place in response to plasmodial infection, providing a new aspect to analyze possible mechanisms of CM. This is the first report on the chemokine expression in neuroglial cells in response to malaria infection.

We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.

Serum levels of interleukin-18 (IL-18), interferon-gamma (IFN-gamma), and immunoglobulin E (IgE) were determined for 96 patients with Plasmodium falciparum malaria admitted to hospital, Bangkok, Thailand in the period 1998-2000. The patients were divided into 3 groups, i.e. uncomplicated, severe and cerebral malaria according to WHO criteria (2000). Elevation of IL-18 levels was observed in all 3 groups, with a tendency for higher levels in cases with severe malaria throughout the course of the disease. Moreover, there was a significant correlation between IL-18 levels and the extent of parasitaemia among patients with severe malaria. However, IL-18 levels decreased more significantly in patients with cerebral malaria compared with the other groups in the late stage of the disease. Elevated levels of IFN-gamma were also observed in all groups of patients, especially in those with severe or cerebral malaria, and the levels in patients with cerebral malaria remained significantly higher than in those with uncomplicated malaria during days 4-7 post-treatment, suggesting the involvement of IFN-gamma in disease severity. Meanwhile, no significant difference was observed in IgE levels between the severe and uncomplicated groups of patients with helminth infection, although IgE levels were significantly higher in helminth-infected patients than uninfected patients. These results suggest that IL-18 plays a key role in inducing severe malaria through another pathway, such as elevation of IFN-gamma, rather than its IgE inducing activity.

The presence of Plasmodium ovale has never been previously reported in Myanmar. Using blood samples obtained in many villages across the country between 1996 and 2000, molecular diagnosis of Plasmodium species was made with semi- or full-nested polymerase chain reaction (PCR) with species-specific primers, followed by agarose gel electrophoresis to detect amplification products. The presence of P. ovale was also confirmed with the another PCR-based diagnosis, the microtiterplate hybridization (MPH) method using species-specific probes. Both methods target the A type of the small subunit ribosomal RNA gene of the four human malaria parasites. Plasmodium ovale DNA was amplified in samples from 65 (4.9%) of 1323 PCR-positive patients, with perfect agreement between results obtained by nested PCR and MPH. Only four P. ovale-infected patients had single-species infection; all others were coinfected with P. falciparum, P. vivax and/or P. malariae. Quadruple infections were observed in six subjects. Parasites with typical P. ovale morphology were found in only 19 patients by conventional microscopy of Giemsa-stained thin smears or fluorescence microscopy of acridine orange-stained thin smears. Plasmodium ovale infections were found in villages situated in the southern, central and western regions of Myanmar, suggesting that P. ovale may be widely distributed in this country.

Sparganum proliferum is a larval cestode for which the adult stage is unknown. It is characterized by the continuous branching and budding when parasitized to humans, and causes fatal human sparganosis. However, the biological features of S. proliferum, including its taxonomic status, still remain obscure. Our previous investigation suggested that S. proliferum might be phylogenetically distinct from Spirometra erinaceieuropaei, by the analysis on mitochondrial NADH dehydrogenase subunit 3 (ND3) gene. However, mitochondrial DNA sequence in Platyhelminth is known to have heteroplasmy within a species. Therefore, in the present study, we have investigated the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene and the partial nucleotide sequences of nuclear coded succinate dehydrogenase iron-sulfur protein subunit gene (sdhB). The results clearly demonstrated that S. proliferum is a distinct species from S. erinaceieuropaei, and that S. proliferum belongs to the order Pseudophyllidea.

Although the strain difference in protection of mice to Schistosoma mansoni infection has been described, limited information is available in the case of Schistosoma japonicum. In the present study, we compared the protective immunity to S. japonicum infection and cytokine production in various strains of mice vaccinated with γ-irradiated cercariae. A significant reduction in worm recovery was observed in male and female mice of DBA/2 at a 6-week interval between vaccination and a challenge infection, whereas vaccinated mice of C57BL/6, C57BL/10, (C57BL/6 × DBA/2) F1 (B6D2F1) and (C57BL/10 × DBA/2) F1 (B10D2F1) showed no detectable level of protection. No sex-linked difference in development of resistance was observed in any of the strains so far examined. Vaccination with γ-irradiated cercariae twice with a 3-week interval also induced significant protection against a challenge infection in DBA/2 but not in BALB/c or C57BL/6 strains. Further studies demonstrated that spleen cells of vaccinated C57BL/6 mice produced lower levels of IFN-γ compared to the cells of vaccinated BALB/c and DBA/2. On the other hand, production of IL-10 by spleen cells was relatively higher in BALB/c mice than in the other two strains. Macrophages that had been stimulated with spleen cell culture supernatants derived from vaccinated DBA/2 damaged schistosomula more effectively than cells stimulated with supernatants derived from the other strains. These results suggest that different levels of protection observed among strains of mice depend on the balance of cytokine responses which consequently activate or suppress macrophage-mediated damage to schistosomula.

The NK cell inhibitory receptor Ly49A recognizes the mouse MHC class I molecule H-2Dd and participates in the recognition of missing self. Previous studies indicated that the determinant recognized by Ly49A exists in α1/α2 domain of H-2Dd. Here we have substituted polymorphic as well as conserved residues of H-2Dd α1/α2 domain (when compared with H-2Kd, which does not interact with Ly49A). We then tested the ability of the H-2Dd mutants to interact with Ly49A by soluble Ly49A tetramer binding and NK cell cytotoxicity inhibition assays. Individual introduction of mutations converting the H-2Dd residue into the corresponding H-2Kd residue (N30D, D77S, or A99F) in H-2Dd partially abrogated the interaction between Ly49A and H-2Dd. Introduction of the three mutations into H-2Dd completely abolished Ly49A recognition. Individual introduction of D29N or R35A mutation into the residues of H-2Dd that are conserved among murine MHC class I severely impaired the interaction. The crystal structure of H-2Dd reveals that D77 and A99 are located in the peptide binding groove and that N30, D29, and R35 are in the interface of the three structural domains of MHC class I: α1/α2, α3, and β2-microglobulin. These data suggest that Ly49A can monitor mutations in MHC class I inside and outside of the peptide binding groove and imply that inhibitory MHC class I-specific receptors are sensitive to mutations in MHC class I as well as global loss of MHC class I. Our results also provide insight into the molecular basis of Ly49A to distinguish MHC class I polymorphism.

By using IL-5 transgenic mice, it has been shown that eosinophils might play a key role in elimination of larval stages of nematode infections. The present study was carried out to clarify molecular mechanisms involved in the eosinophil-mediated killing of Nippostrongylus brasiliensis larvae. The larvicidal activity was observed in the presence of normal serum in vitro. Electron microscopic observations revealed firm attachment of eosinophils to the cuticular surface of larvae, which was damaged by electron-dense materials released from eosinophils. The larvicidal activity was abrogated by heat- or zymosan-treatment of the serum, whereas depletion of IgG or IgM from the serum did not interfere with eosinophil adhesion and killing. Moreover, pretreatment of eosinophils with monoclonal antibodies against CD11b or VLA-4 inhibited the eosinophil-mediated killing of larvae. Immunofluorescent staining demonstrated the deposition of C3c and plasma fibronectin on the cuticle of the larvae. These results indicate that interactions between CD11b and VLA-4 and their respective counter-ligands deposited on the cuticle are essential in eosinophil-mediated adhesion and damage to larvae of N. brasiliensis.

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

Paramyosin (PM), a myosin-like protein is a major antigen on Schistosoma japonicum (Sj). We reported that passive transfer of a monoclonal IgE SjE18 e .1 which recognizes PM of Sj (SJPM), partially protected mice from challenge infection. In the present study, we developed an experimental model system of schistosomiasis japonica with domestic pigs in China and used it for the evaluation of vaccination with recombinant SJPM (rSJPM). Sixteen-week-old pigs were successfully infected by dermal penetration of 120 cercariae of a domestic strain of Sj (50–60% worm recovery 11 weeks after challenge). The pigs vaccinated with 400 UV attenuated cercariae showed a reduction of worm recovery (53%, p

Complex II of adult Ascaris suum muscle exhibits high fumarate reductase (FRD) activity and plays a key role in anaerobic electron-transport during adaptation to their microaerobic habitat. In contrast, larval (L2) complex II shows a much lower FRD activity than the adult enzyme, and functions as succinate dehydrogenase (SDH) in aerobic respiration. We have reported the stage-specific isoforms of complex II in A. suum mitochondria, and showed that at least the flavoprotein subunit (Fp) and the small subunit of cytochrome b (cybS) of the larval complex II differ from those of adult. In the present study, complete cDNAs for the iron-sulfur subunit (Ip) of complex II, which with Fp forms the catalytic portion of complex II, have been cloned and sequenced from anaerobic adult A. suum, and the free-living nematode, Caenorhabditis elegans. The amino acid sequences of the Ip subunits of these two nematodes are similar, particularly around the three cysteine-rich regions that are thought to comprise the iron-sulfur clusters of the enzyme. The Ip from A. suum larvae was also characterized because Northern hybridization showed that the adult Ip is also expressed in L2. The Ip of larval complex II was recognized by the antibody against adult Ip, and was indistinguishable from the adult Ip by peptide mapping. The N-terminal 42 amino acid sequence of Ip in the larval complex II purified by DEAE-cellulofine column chromatography was identical to that of the mature form of the adult Ip. Furthermore, the amino acid composition of larval Ip determined by micro-analysis on a PVDF membrane is almost the same as that of adult Ip. These results, together with the fact, that homology probing by RT-PCR, using degenerated primers, failed to find a larval-specific Ip, suggest that the two different stage-specific forms of the A. suum complex II share a common Ip subunit, even though the adult enzyme functions as a FRD, while larval enzyme acts as an SDH.

We report the first case of rectal carcinoma associated with S. japonicum and membranous nephropathy. A 57-year-old Japanese man noticed narrowing of his feces. He had lived in Yamanashi prefecture, an endemic area of S.japonicum. He had suffered from nephrotic syn­ drome for about 1 year. Barium enema study showed a severe stricture in the upper rectum and biopsy specimens from the tumor demonstrated well differentiated adenocarcinoma and many ova of S. japonicum. Sonography of the liver showed a network pattern and a linear high echoic area. Low anterior resection with incisional biopsy of the liver and the right kidney was performed. Histopathological findings showed well differentiated adenocarcinoma and schisto­ somal ova. The total number of ova in the resected colon amounted to 15 133, consisting of 2243 inside and 12 890 outside the carcinoma. The nearer to the carcinoma the area was, the higher was the density of ova. The findings of light microscopy and electron microscopy of the biopsy specimen from the kidney were compatible with membranous nephropathy (stage II). This case suggests that schistosomal ova have some effect on carcinogenesis and nephrotic syndrome. In patients with nephrotic syndrome of unknown cause, especially in inhabitants of endemic areas of S. japonicum, gastrointestinal malignancy should be ruled out as an etiolog­ ical factor. Sigmoidoscopy would be useful for colorectal carcinoma surveillance in S. japoni­ cum patients.

A rapid single-step screening method for detection of glucose-6-phosphate dehydrogenase (G6 PD) deficiency was evaluated on Halmahera Island, Maluku Province, Indonesia, and in Shan and Mon States, Myanmar, in combination with a rapid diagnosis of malaria by an acridine orange staining method. Severe deficiency was detected by the rapid test in 45 of 1126 volunteers in Indonesia and 54 of 1079 in Myanmar, but it was difficult to distinguish blood samples with mild deficiency from those with normal activity. 89 of 99 severely deficient cases were later confirmed by formazan ring method in the laboratory, but 5 with mild and 5 with no deficiency were misdiagnosed as severe. Of the samples diagnosed as mild and no deficiency on-site, none was found to be severely deficient by the formazan method. Malaria patients were simultaenously++ detected on-site in 273 samples on Halmahera island and 277 samples from Shan and Mon States. In Mon State, primaquine was prescribed safely to G6 PD-normal malaria patients infected with Plasmodium vivax and/or gametocytes of P. falciparum. The new rapid test for G6 PD deficiency may be useful for detecting severe cases under field conditions, and both rapid tests combined are can be useful in malaria-endemic areas, facilitating early diagnosis, prompt and radical treatment of malaria and suppression of malaria transmission.

Diagnosis and vaccine development form the major focus in creating strategies for the control of schistosomiasis. In this study, we established an IgG1 mouse monoclonal antibody (MoAb), SJA111, which strongly reacted with 23–25-kDa Schistosoma japonicum tegumental-associated membrane proteins, but not with eight other parasitic antigens. A λgt 11 cDNA library from the Japanese strain of the Schistosoma japonicum adult worm was screened with SJA111 as a probe. A single positive clone was isolated and the nucleotide sequence of the isolated cDNA was determined. The cDNA clone consisted of 844 bp, and the coding region contained 576 bp which was translated to a 22.6-kDa protein. This region showed 99.0% and 99.3% significant homology with those of the Chinese and Philippine strains of Schistosoma japonicum , respectively. The deduced amino acid sequence of the protein was identical to that of the Philippine strain and only one residue differed from that of the Chinese strain. The recombinant form of the tegumental protein was expressed in Escherichia coli and purified by a combination of ion exchange and affinity chromatography, and the purified protein was found to react with the sera of patients infected with Schistosoma japonicum . This result suggests that this antigen may be useful in the immunodiagnosis of schistosomiasis as well as in the development of an effective vaccine.

The sensitivity of a dipstick-ELISA for urinary schistosomiasis was evaluated alongside microscopy. Out of 155 subjects screened, 28 (18.1%) had symptoms (hematuria and/or painful micturition) and 12 (42.8%) were microscopically positive for S. haematobium eggs, even though three (25%) were confirmed upon repeated examination. The dipstick-ELISA detected 11/12 (91.7%) of them at first examination and missed one which was nevertheless positive on subsequent tests. Of 127 asymptomatic individuals 14 (11%) had eggs although eight (57.1%) were identified by up to eight subsequent examinations. The dipstick, however, detected all 14 cases at first screening together with 10 others that remained egg negative thereby suggesting higher absolute sensitivity. In a longitudinal evaluation of prevalence using a cohort of infected individuals the dipstick-ELISA had an average prevalence of 92% compared to 70.2% by microscopy. Indeed only the dipstick-ELISA ever estimated the true prevalence of 100% showing that it was relatively more sensitive.

Sparganum proliferum is characterized by continuous branching and budding, the resulting progeny invading all tissues of the human body, causing fatal sparganosis. Its life cycle, definitive hosts and the route of infection to humans have not yet been disclosed. Because its morphology is similar to Spirometra erinacei, the phylogeny of S. proliferum has been thought to be identical to or closely related to S. erinacei. However, the taxonomy of S. proliferum has not been established up to present due to the lack of definitive observations. In order to clarify the phylogenetic relationship between S. proliferum and S. erinacei, nucleotide sequences of mitochondrial NADH dehydrogenase subunit 3 gene (ND3) and four mitochondrial tRNA coding genes of S. proliferum and other pseudophyllidean cestodes were analyzed. The sequences of S. proliferum showed high similarity to those of S. erinacei, although they were clearly different from each other, indicating that the phylogeny of S. proliferum and S. erinacei is distinct. This is the first report showing the phylogenetic relationship among S. proliferum and other pseudophyllidean cestodes at the DNA sequence level.

Young adults of the squirrel monkey, Saimiri sciurea, were immunized with γ-irradiated (20 krad) cercariae of Schistosoma joponicum, and they were examined for the protection against a challenge infection with normal cercariae. In the unimmunized control group, dominant accumulation of neutrophils was observed in egg granulomas in the liver, intestine, and lungs in association with severe hemorrhage in surrounding tissues. In the immunized group, on the other hand, the granulomas were smaller in size and number, and infiltration of lymphocytes and eosinophils within the granulomas was observed as a conspicuous cellular response. Moreover, there was no hemorrhage in the surrounding tissues in immunized animals, suggesting that the vaccination with attenuated cercariae was effective in reducing pathologic damages in the involved tissues, even though there was no significant reduction in the worm recovery in these animals compared with unimmunized controls. After the challenge infection, immunized animals demonstrated earlier and higher IgG antibody responses against egg and adult worm antigens. These results indicate that the squirrel monkey provides a useful vaccine model for S. joponicum infection. As far as we know, this is the first report on S. joponicum infection in this species.

Nippostrongylus brasiliensis infection is characterized by blood and tissue eosinophilia induced by interleukin (IL)-5 secreted from CD4+ T cells. However, it is still obscure whether eosinophils play an important role in the protection against N. brasiliensis infection. In this study we attempted to determine whether the in vivo environment of IL-5 transgenic mice, characterized by high eosinophil production, could affect the worm burden after N. brasiliensis infection. Kinetic studies on the infection demonstrated a significantly lower worm recovery from the intestine of IL-5 transgenic mice compared to age-matched background controls. This tendency was also observed at the lung stage of the infection. Furthermore, with respect to elevation of the serum IgE concentration, the peak level was observed at 2 weeks after infection in infected background control mice with four times higher concentrations than those of uninfected mice. In contrast, the increase of IgE concentration in IL-5 transgenic mice was very limited and low. The adoptive transfer of eosinophils from IL-5 transgenic mice into background control animals resulted in the reduction of worm recovery from the lungs, suggesting that eosinophils play a key role in the protection against migrating larvae of N. brasiliensis. These results indicate that the innate high level of eosinophils due to constitutive production of IL-5 augments immunity against N. brasiliensis infection.

A rapid, visually read monoclonal antibody (MoAb)-based dipstick assay for specific diagnosis of urinary schistosomiasis was field tested with microscopy and the use of hematuria and proteinuria in a schistosomiasis hematobia endemic area in Southern Ghana. The study group consisted of 229 individuals (114 males and 115 females) aged 1 to 86 years; 145/229 (63.3%) of the subjects submitted stool samples from which no S. mansoni eggs were detected. However, infections with Necator americanus (hookworms) 33.1%, Ascaris lumbricoides 2.8%, Trichuris trichiura (whipworm) 2.8%, and Strongyloides stercoralis 0.7% were detected but did not appear to influence the results of the MoAb-dipstick assay. Urinary schistosomiasis prevalence was estimated as 47.6% by microscopy, 48% by MoAb-dipstick, 39.7% by microhematuria, and 23.6% by proteinuria. The MoAb-dipstick correctly identified 108/109 (99.1%) of microscopically confirmed cases and 118/120 (98.3%) of egg-negative individuals, thereby giving a sensitivity of 99.1% and a specificity of 98.3%. On the other hand, microhematuria and proteinuria were, respectively, 76.1% and 40.4% sensitive, and 94.2% and 92.5% specific when compared to microscopy. Microhematuria and proteinuria had significantly lower sensitivity (P < 0.001) than either microscopy or dipstick.

The AnaeroPack® malaria culture system with a portable thermostat incubator was evaluated in a field laboratory on the Thai-Myanmar border conducting in vitro drug susceptibility tests on blood samples from 5 Karen children infected with P. falciparum. Only one isolate was susceptible to chloroquine; the others were highly resistant. The IC50 value of an isolate was only resistant to mefloquine, whereas the values of the 3 patients who presumably showed recrudescence were slightly elevated in the susceptible ranges. These results suggested that chloroquine should no longer be used for P. falciparum malaria in this geographic area, and that mefloquine should be carefully monitored for its in vivo effectiveness. In this study, the AnaeroPack® malaria culture system with portable thermostatic incubator is a powerful and useful mobile tool, which aids in providing detailed evidence-based distribution data concerning of drug resistant malaria in the field.

p50 is phosphorylated in mitogen-stimulated T cells, and translocated from the membrane to the cytosol after activation of protein kinase C. Sequence analysis of p50 revealed that it is identical with LSP1, a putative calcium-binding and actin-binding protein. lymphocyte form of p50 exhibits heterogeneity in the apparent molecular mass on SDS-PAGE, 50 and 52 kDa (pp50 and pp52), and each isoform exhibits heterogeneity in the isoelectric point, when examined by two-dimensional PAGE. When the two molecular mass variants of p50 were dephosphorylated with alkaline phosphatase, both isoforms showed the same apparent molecular mass of 50 kDa on SDS-PAGE, but could be distinguished by their distinct isoelectric points. Dephosphorylated pp50 (p50a) has an acidic pI compared with dephosphorylated pp52 (p50b). Comparison of the peptide maps of purified p50a and p50b on HPLC revealed that the difference was limited to one peptide peak. NH2-terminal sequence and mass spectrometric analyses of these peptides showed that the peptides derived from p50a and p50b had the same NH2-terminal amino acid sequence up to eight residues, but had distinct molecular masses, 5,533.4 and 6,318.6 Da, respectively. These data suggested that pp52 (p50b) is the product of the previously cloned cDNA and the reduction in the molecular mass of the p50a-derived peptide could be explained by deletion of six amino acid residues, EHLIRH or HLIRHQ.(ABSTRACT TRUNCATED AT 250 WORDS)

A lymphocyte-specific protein, p50, is phosphorylated on Ser and Thr residues in mitogen-activated T cells, suggesting that this molecule plays some role in the T cell activation cascade. p50 was identified as lymphocyte specific protein 1 (LSP1), which is a putative calcium-binding protein. In the present study, to clarify the role of p50 protein in the cascade, in vivo and in vitro phosphorylation of this molecule, and the effect of the phosphorylation on its distribution in activated T cells were examined. First, to obtain a sufficient amount of p50 as a phosphorylation substrate, p50 cDNA, which encodes a protein of 330 amino acid residues with a molecular mass of 36,728 Da, was cloned from an ICR mouse thymocyte cDNA library and expressed in Escherichia coli. When the putative coding region of p50 cDNA was expressed in E. coli, the product showed an apparent molecular mass of 50 kDa on SDS-PAGE. The recombinant p50 was phosphorylated in vitro by rabbit protein kinase C (PKC) and by murine cytosolic protein kinase, that was activated by a combination of phosphatidylserine and diacylglycerol. Furthermore, p50 was shown to be phosphorylated on the same sites in T cells upon stimulation with Con A as when phosphorylated in vitro by rabbit PKC, indicating that p50 is phosphorylated by PKC in Con A-stimulated T cells. On subcellular fractionation followed by immunoblotting analysis, membrane-bound p50 was shown to be released from the membrane following activation of PKC in T cells. These results and the recent finding that p50 binds to actin fibers raise the possibility that p50 controls the binding of actin fibers to the plasma membrane under regulation by PKC in T cells.

The sensitivity of Plasmodium vivax to chloroquine in vitro was investigated in patients admitted to the Bangkok Hospital for Tropical Diseases, Thailand, between September 2001 and May 2002. Of 42 isolates, 34 were successfully tested for parasite sensitivity to chloroquine in vitro; the results showed a significant decrease in sensitivity compared with data published in 1989 and 1995: the IC50 and IC90 were 187.2 and 1217.9 ng/mL blood, respectively, an approximate 4-fold decrease in sensitivity in comparison with other data from the past 2 decades. A number of in vitro tests were performed simultaneously using both WHO microplates and our own laboratory-prepared pre-dosed microplates under the same conditions and there was no significant difference between the results.

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle, and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms. In this study, homology probing with mixed primers for the polymerase chain reaction and subsequent sequence analysis were successfully applied to clone cDNA for the flavoprotein (Fp) subunit of human liver complex II. The isolated clone contains an open reading frame of 1,992 nucleotides and encodes a mature protein of 621 amino acids with a molecular weight of 68,011. The amino acid sequence was highly homologous with that of bovine heart Fp (93.2%) and was quite different from the partial sequence of human placental Fp reported previously [Malcovati et al. (1991) in Flavins and Flavoproteins 1990, pp. 727-730], which showed striking homology to that of Bacillus subtilis. To solve this discrepancy, the partial cDNA sequences of the stomach and placental Fp subunits of human complex II were determined in addition to the full length cDNA of liver. The sequence data, sensitivity to thiol reagents and antigenic properties indicated that the major from of FP subunit in human complex II is unique at least among the three tissues analyzed, and is more similar to the Fp subunit of bovine heart than to that of B. subtilis.

Participation of IgE in protective immunity against Schistosoma japonicum was examined by comparing congenital IgE-deficient SJA/9 and IgE-producing SJL/J mice. Mice immunized with 100 irradiated cercariae 7 weeks previously were infected with 50 live cercariae. In SJL/J mice at 40 days after infection, a 3-to 4-fold increase of total IgE levels and anti-S, japonicum egg IgE antibody production were observed with no significant difference between immunized and nonimmunized mice. IgE was not detected in SJA/9 mice throughout the experiments. Protective immunity evaluated by recovery of adult worms was found in SJA/9 mice and was comparable to that of SJL/J mice. These results suggest that acquired immunity in mice with irradiated cercariae of S. japonicum was not dependent on IgE in these strains of mice.

Human recombinant tumour necrosis factor (rTNF) enhanced monoclonal IgE-dependent eosinophil-mediated cytotoxicity to schistosomula of Schistosoma japonicum in a dose-dependent manner. The enhancing effect of rTNF was also observed for the antibody-dependent cytotoxicity of a human eosinophilic leukemia cell line, EoL-3, but not of another cell line, EoL-1. Observation by a slow-motion movie camera demonstrated that activated EoL-3 cells adhered to the surface of schistosomula by 6 h after incubation, which triggered intracellular movement of eosinophil granules. The granules were concentrated toward the surface of the larvae and then degranulation started. The cell membrane was left as a balloon-like remnant. Cell sorting analysis by FACStar indicated that the expression of receptors for C3bi (CR3) and low affinity FcR for IgE (Fc epsilon RII) increased on the surface of EoL-3 cells after stimulation with rTNF, while this was not observed for EoL-1 cells.

Homology probing by using mixed primers for polymerase chain reaction (PCR) and a subsequent sequence analysis by automated DNA sequencer were applied to determine a partial cDNA sequence of the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase). Complex II is a membrane-bound flavoenzyme, which catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle, and it is a component of the mitochondrial and bacterial respiratory chains. In this study, the partial amino acid sequence of iron-sulfur subunits in Caenorhabditis elegans mitochondria was deduced from the DNA sequence obtained from cDNA-PCR. Mixed oligonucleotide primers corresponding to two conserved regions which appear to be the binding site for the prosthetic group were used. The product of PCR was cloned into plasmid vector pUC 119 and the sequence was determined from double strand plasmid DNA by the dideoxy method using of one-dye, four-lane type the automated DNA sequencer (DSQ-1, Shimadzu). The PCR product contained 483 nucleotides and its deduced amino acid sequence was highly homologous with that in human liver (68.9%) and that of Escherichia coli sdh B product (50.3%). As expected, striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme.

Passive transfer of immunity using a mouse monoclonal IgE antibody against Schistosoma japonicum was found to be enhanced by heterologous helminth infections. BALB/c mice were infected with Toxocara canis or Nippostrongylus brasiliensis so as to induce eosinophilia prior to a challenge infection with S. japonicum. Recovery of adult schistosomes decreased in a group of mice that had been infected with T. canis and challenged with cercariae at the cutaneous site of sensitization with the IgE antibody as compared with that in mice that had been similarly treated with normal serum in the absence of T. canis infection. Histological examinations revealed a close association of polymorphonuclear cells, including eosinophils, with damaged schistosomula in the skin of T. canis-infected mice that had received the IgE antibody. An enhancement in worm reduction was also observed in mice harboring either of both nematodes when the monoclonal antibody had been injected intraperitoneally during the phase of migration of schistosomula from the skin to the lungs. In vitro studies on macrophage-mediated damage to schistosomula suggested that the enhancement in worm reduction was at least partly due to the activation of macrophages induced by the heterologous infections.

Nan Province, located in northern Thailand, is hyperendemic for parasite infections; the helminthic infection rate in 1,010 schoolchildren was 60.0% in 2001. Mass anthelmintic chemotherapy has been conducted with schoolchildren, and selective treatment has been given to people in the community, from 2002. The modified cellophane thick smear method was used to examine the prevalence and intensity of helminth infections in schoolchildren and community people once a year during the period 2002-2004. The prevalence of helminth infections decreased slowly from 60.0 to 40.3% in schoolchildren and from 70.8 to 60.0% in the older age population. Three parasite species were common: hookworm, Ascaris and Haplorchis, an intestinal trematode. Hookworm presented throughout the whole district. Ascaris infection occurred at high rates in some villages, while in some villages none was found. The villages where Ascaris infection was nil had high rates of Haplorchis infection, and vice versa. Most hookworm and Trichuris infections were of light intensity. Heavy intensity infection was found in 12.8-18.1% of Ascaris cases examined. Parasite infection rates in Chaloem Phra Kiat District can be classified as low prevalence.

Saraburi Province, Central Thailand has been a paragonimiasis-endemic area since 1956. This study compared the prevalences of human paragonimiasis in two villages near Chet Khot Waterfall, Kaeng Khoi District, investigated in 1984-1985 and 2005. The results from the 1980s showed 6.3% and 1% of villagers were positive for Paragonimus eggs in sputum and stool, respectively. In 2005, Paragonimus eggs were not found in feces or sputum. An IgG-ELISA for paragonimiasis was conducted on 33 serum samples collected in the 1980s, 23 collected in 2005 and 25 diagnosed with other parasitic infections. Ninety percent of the samples from the eighties were positive for paragoimiasis, and 43% from 2005 were positive, equivalent to 10.9% and 4.9% of the total population examined in the 1980s and 2005, respectively. Serodiagnosis is currently the best method for detecting paragonimiasis. The positive cases in the 1980s were age 10-60 years and in 2005 were age 34- 67-years-old. The prevalence and intensity of Paragonimus metacercariae in fresh Waterfall crabs collected from Chet Khot Waterfall were significantly lower in the 1980s than in 2005. The prevalence of paragonimiasis in this endemic area has decreased to the level that no egg-producing cases were detected. No infections were found in villagers age30 years, despite the high density of metacercariae in the crabs, indicating a change in the habit of eating raw food among the younger people.

ACIPAC has made an effort to promote the concept of the school-based approach to malaria and STH control, mainly through human resource development, which could be eventually extended to any other health promotion program. Implementation of SSPP resulted in the establishment of national policies on parasite control and/or school health in some partner countries. It also provides a good opportunity for the formulation of partnerships among health and education sectors and international partners, although it did cause some problems concerning the enrollment of persons of authority from partner countries, and the staff of JICA resident offices as well. As described in the Joint Evaluation Report, ACIPAC is expected to further contribute to human resource development and to strengthening human resource and information networking at regional and global levels.

Japan controlled its major parasitic diseases by the 1970s. Based on this experience, the Government of Japan proposed the Global Parasite Control Initiative in 1998 and established three research and training centres around the world. The Asian Centre of International Parasite Control (ACIPAC) is the first such centre, and completed five years of activities focused on school-health-based parasite control in the Greater Mekong Subregion in 2005. The lessons learned and experiences gained by ACIPAC should be applied to all health promotion programmes worldwide.