Surgical treatment of hepatic cystic echinococcosis in patients co-infected with HIV/AIDS.

Co-infections of cystic echinococcosis (CE) and HIV/AIDS is rare. We report four CE cases that were HIV positive. Three out of the four patients underwent a surgical operation to remove the hydatid cysts in their livers. The operation confirmed that in two of the cases their cysts had ruptured. These patients were given 3 months of albendazole after the operation. Follow-up showed they were remarkably improved in term of their health, although they were still HIV antibody positive 6 months after surgical treatment. Interestingly, the treatment remarkably increased their CD4+ cell population. We showed that surgery is suitable for treating hepatic cystic echinococcosis with HIV/AIDS co-infection.

Immunodiagnosis of sheep infections with Echinococcus granulosus: in 35 years where have we come?

W. K. Yong and D. D. Heath published in 1979 a seminal paper in the first issue of Parasite immunology describing their efforts to determine whether the arc 5 precipitin band, formed when test human serum is reacted against electrophoresed hydatid cyst fluid antigen, would be a suitable immunodiagnostic test for the identification of sheep infected with Echinococcus granulosus. Although they found antibodies to arc 5 in the sera of hydatid-infected sheep, the sera of some sheep harbouring Taenia ovis and T. hydatigena also precipitated the hydatid cyst fluid arc 5 antigen, so they concluded arc 5 antibodies were not suitable for the specific immunodiagnosis of E. granulosus infection in sheep in New Zealand. Subsequent work has shown that the existence of multiple infections with different taeniid species, antigenic cross-reactivity between these related parasites and the low level of specific antibody response to infection continue to hinder efforts to improve the diagnosis of hydatid infection in sheep and other natural intermediate hosts, thereby preventing the development of any practical test. In particular, the poor antibody response of ruminants to naturally acquired hydatid infection may prove an insurmountable barrier in future efforts to develop a reliable and accurate immunological test.

Current status of the genetics and molecular taxonomy of Echinococcus species.

The taxonomy of Echinococcus has long been controversial. Based mainly on differences in morphology and host-parasite specificity characteristics, 16 species and 13 subspecies were originally described. Subsequently, most of these taxa were regarded as synonyms for Echinococcus granulosus and only 4 valid species were recognised: E. granulosus; E. multilocularis; E. oligarthrus and E. vogeli. But, over the past 50 years, laboratory and field observations have revealed considerable phenotypic variability between isolates of Echinococcus, particularly those of E. granulosus, which include differences in: morphology in both larval and adult stages, development in vitro and in vivo, host infectivity and specificity, chemical composition, metabolism, proteins and enzymes, pathogenicity and antigenicity. The application of molecular tools has revealed differences in nucleic acid sequences that reflect this phenotypic variation and the genetic and phenotypic characteristics complement the previous observations made by the descriptive parasitologists many years ago. The fact that some of these variants or strains are poorly or not infective to humans has resulted in a reappraisal of the public health significance of Echinococcus in areas where such variants occur. A revised taxonomy for species in the Echinococcus genus has been proposed that is generally accepted, and is based on the new molecular data and the biological and epidemiological characteristics of host-adapted species and strains.

Novel immunomic technologies for schistosome vaccine development.

Schistosomiasis remains one of the most common human helminthiases, despite the availability of an effective drug against the causative parasites. Drug treatment programmes have several limitations, and it is likely that a vaccine is required for effective control. While decades of vaccine development have seen the discovery and testing of several candidate antigens, none have shown consistent and acceptable high levels of protection. The migrating larval stages are susceptible to immunity, however few larval-specific antigens have been discovered. Therefore, there is a need to identify novel larval-specific antigens, which may prove to be more efficacious than existing targets. Immunomics, a relatively new field developed to cope with the recent large influx of biological information, holds promise for the discovery of vaccine targets, and this review highlights some immunomic approaches to schistosome vaccine development. Firstly, a method to focus on the immune response elicited by the important and vulnerable larval stage is described, which allows a targeted study of the immunome at different tissue sites. Then, two high-throughput arrays are discussed for the identification of protein and carbohydrate antigens. It is anticipated that these approaches will progress vaccine development against the schistosomes, as well as other parasites.

SerpinB2 deficiency modulates Th1/Th2 responses after schistosome infection.

SerpinB2, also known as plasminogen activator inhibitor type-2, is a major product of macrophages and is upregulated during many infections. Although SerpinB2 inhibits urokinase plasminogen activator in vitro, evidence that this represents its physiological role in vivo is not compelling. We have recently shown that SerpinB2-/-mice generate enhanced Th1 responses after immunization with a Th1 immunogen. Herein,we show that Schistosoma japonicum granulomas induced liver SerpinB2 mRNA expression by >600-fold in wild-type mice. In SerpinB2-/- mice, worm and egg burden, and granuloma number and volume were unaffected. However, granulomas in these mice were associated with reduced fibrosis (as determined by Sirius red staining and image analysis) and increased iNOS, IL-6, IL-10 and TNFa and decreased Arg 1 and IL-13 mRNA expression. SerpinB2-/- mice immunized with soluble egg antigen (SEA) also showed reduced levels of SEA-specific IgG1. SerpinB2 deficiency thus promoted certain Th1 and reduced certain Th2 responses in response to this Th2 immunogen.

Immunopathogenesis of human schistosomiasis.

Schistosomiasis continues to be a significant cause of parasitic morbidity and mortality worldwide. This review considers the basic features of the pathology and clinical outcomes of hepatointestinal and genitourinary schistosomiasis, presents an overview of the numerous studies on animal models that have clarified many of the immunopathological features, and provides insight into our current understanding of the immunopathogenesis and genetic control of human schistosomiasis. In murine schistosomiasis, pathology is induced by a CD4(+) Th2 driven granulomatous response directed against schistosome eggs lodged in the host liver. The Th2 cytokines IL-4 and IL-13 drive this response, whereas IL-10, IL13Ralpha2, IFN-gamma and a subset of regulatory T-cells act to limit schistosome induced pathology. A variety of cell types including hepatic stellate cells, alternatively activated macrophages and regulatory T-cells have also been implicated in the pathogenesis of schistosomiasis. Current knowledge suggests the immunopathogenic mechanisms underlying human schistosomiasis are likely to be similar. The review also considers the future development of anti-pathology schistosome vaccines. As fibrosis is an important feature of many other diseases such as Crohn's disease and sarcoidosis, a comprehensive understanding of the cellular and molecular mechanisms involved in schistosomiasis may also ultimately contribute to the development an effective disease intervention strategy for other granulofibrotic diseases.

Reflections on the biochemistry of Echinococcus: past, present and future.

This review discusses 5 of my earliest papers on the biochemistry of larval Echinococcus published in Parasitology in the 1970s and 1980s. Two of the publications consider aspects of the basic biochemistry, intermediary metabolism and the regulation of respiratory pathways in E. granulosus and E. multilocularis, and emphasize the existence of inter- and intra-species variation in their general metabolism. The third reports on the detailed biochemical analysis of the tegumental surface of the protoscolex of E. granulosus, and the final 2 papers describe the genomic cloning of Echinococcus DNA fragments and their use, along with other DNA markers, in molecular identification of E. granulosus isolates collected worldwide from areas endemic for hydatid disease. A number of years have elapsed since these publications in Parasitology and, in this Centenary Issue article, I reflect briefly on some of the subsequent studies undertaken in these research areas that have advanced the field. As well, I provide brief insight on new research directions, emphasizing the impact of molecular biology and associated techniques on future studies of Echinococcus and hydatid disease.

Familial aggregation of human helminth infection in the Poyang lake area of China with a focus on genetic susceptibility to schistosomiasis japonica and associated markers of disease.

Human helminthiases are common in China, especially in rural areas where sanitation conditions are poor. Soil-transmitted helminths (STHs) are predominantly found in the southern provinces. Schistosoma japonicum is also endemic to southern China. Here we review the prevalence of helminth infections and polyparasitism in China, and discuss the interactions between helminth parasites in the co-infected host. It is clear that STHs are more prevalent in rural China than previously suggested emphasizing the need for systematic control of STHs. Further, the need for improved sanitation and hygiene conditions to prevent parasite transmission is highlighted. We provide supporting evidence for human genetic susceptibility to both single helminth infection and polyparasitism, and suggest that susceptibility to helminths infections may not be independent of one or the other. We demonstrate an association between single nucleotide polymorphism (SNP) variants in IL-5 and symptomatic S. japonicum infection and discuss the potential role of IL-5 in other helminth infections. Fundamental to disease and morbidity control is adequate and effective diagnosis and surveillance of disease. We discuss the role of sICAM-1 and TNFR-I and -II as candidate markers for schistosome-induced hepatomegaly and fibrosis, and their potential for assessing disease stage and progression in schistosomiasis.

Comparative real-time PCR and enzyme analysis of selected gender-associated molecules in Schistosoma japonicum.

Schistosomes are complex parasitic helminths with discrete life-cycle stages, adapted for survival in their mammalian and snail hosts and the external aquatic environment. Recently, we described the fabrication and use of a microarray to investigate gender-specific transcription in Schistosoma japonicum. To address transcriptional differences, 8 gender-associated gene transcripts identified previously by the microarray analysis were selected for further study. First, differential transcription patterns were investigated in 4 developmental stages using real-time PCR. Subsequently, we undertook functional analysis of a subset of 4 transcripts encoding metabolic enzymes, so as to correlate gender-associated transcript levels with enzyme activity in protein extracts from adult worms. The 8 characterized molecules serve as a basis for further investigation of differential gene expression during the schistosome life-cycle and for studying the sexual dimorphism of adult worms. Continual refinement and annotation of the microarray used in the current study should support future work on these aspects.

Analysis of the cox1 gene in Echinococcus granulosus from sheep in northeast Iran using PCR high-resolution melting (qPCR-HRM) curve analysis.

Echinococcus granulosus, the etiologic agent of echinococcosis, is one of the most important zoonotic helminthes worldwide. Knowledge of E. granulosus species and genotypes has important implications for epidemiology, control, and prevention of diseases as well as future vaccine and drug designs. There are many molecular methods developed to define genotypes of E. granulosus, among them high resolution melting (HRM) analysis, as a new approach, is a single step and closed tube method. It is appropriate for fast screening of large number of isolates. This technique is an accurate, user friendly, cost-effective, fast and simple method, which does not need post-PCR processes. Between March and lst august 2016, of 726 sheep examined in abattoirs in Razavi Khorasan province, Northeast Iran, 109 harboured cystic echincoccosis lesions (liver samples= 65 and lung samples= 44) which were collected for analysis. Total genomic DNA was extracted from each sample and amplified for the presence of polymorphism in the mitochondrial cox1 gene of Echinococcus granulosus using a high resolution melting curve (HRM) method. A total of 109 hydatid cyst samples analyzed by PCR high-resolution melting (qPCR-HRM) curve of the cox1 gene, all isolates were identified as G1 genotype (sheep strain). G1 is the predominant genotype in sheep in northeast of Iran. The high incidence of the G1 genotype (known to be the predominant E. granulosus genotype infecting humans globally) in sheep has considerable implications for hydatid disease control programs in this area.

Molecular confirmation of a case of multiorgan cystic echinococcosis.

We report on the results of radical surgery performed on a 10-yr-old Chinese female with multiple echinococcosis lesions and the diagnosis of the infection by imaging, histology, serology, and DNA analysis. Molecular genotyping provided unequivocal proof that the patient was infected with Echinococcus granulosus, the cause of cystic echinococcosis.

A leucine zipper protein of mitochondrial origin.

Sequence-specific DNA-binding proteins are characterised by short coiled-coil structural domains classified as zinc finger/RING finger, leucine zipper (L-Zip) or helix-loop-helix (HLH) motifs. The L-Zip proteins are defined by a pattern of at least four leucine (L) residues repeated every seventh amino acid that mediates protein dimerisation through the formation of parallel alpha-helical dimers. Usually the zipper is incorporated into a helix-loop-helix conformation called the basic helix-loop-helix-leucine zipper (bHLH/Zip). To date, all of the several hundred proteins reported as containing the L-Zip and/or bHLH/Zip motifs are nuclear-encoded. No leucine zipper polypeptide has, hitherto, been reported as mitochondrial in origin. Here we report such a polypeptide, the nicotinamide dehydrogenase subunit 4L (nad4L). We first identified this in human blood flukes of the genus Schistosoma (phylum Platyhelminthes; class Trematoda) but show that this is a common feature in other eucaryotes as well. Therefore, in addition to their well recognised role in oxidative phosphorylation, nad4L proteins may be pivotally involved in a range of other biological processes such as transcription and/or replication activation or as signal transmitters in communication with the nucleus and other cellular organelles. This may indicate a link between transcription regulation and respiration in mitochondria. We have also identified L-Zip-like motifs in nuoK, the procaryotic equivalent of the nad4L mitochondrial protein.

Sjalpha elements, short interspersed element-like retroposons bearing a hammerhead ribozyme motif from the genome of the oriental blood fluke Schistosoma japonicum.

Smalpha is a short interspersed element (SINE)-like retroposon that occurs in high copy number of the genome of the human blood fluke Schistosoma mansoni. The sequence of the consensus Smalpha element includes the hallmark features of SINE-like elements including a promoter region for RNA polymerase III, an AT-rich stretch at its 3'-terminus, a short length of 500 bp or less, and short direct repeat sequences flanking the insertion site. Interestingly, the sequence of Smalpha also encodes an active ribozyme bearing a hammerhead domain. Contrary to the recent findings of Ferbeyre et al. (Mol. Cell. Biol. 18 (1998) 3880-8) that indicated that Smalpha-like elements were absent from the genome of the Oriental blood fluke Schistosoma japonicum, we report here that the genome of S. japonicum does contain a family of Smalpha-like retroposons, elements that we have named the Sjalpha family. Like Smalpha, Sjalpha elements are SINE-like in structure and sequence, are present at high copy number interspersed throughout the S. japonicum genome, and contain an ostensibly functional, hammerhead ribozyme motif. The presence of these elements in all species of Schistosoma so far examined suggests that the hammerhead domain was acquired by vertical transmission from a common schistosome ancestor.

Molecular and immunological characterisation of a polymorphic cytosolic fatty acid binding protein from the human blood fluke of humans, Schistosoma japonicum.

Most organisms obtain their fatty acids through their diet or by de novo synthesis, but human blood flukes belonging to the genus Schistosoma lack the oxygen-dependent pathways required for the synthesis of sterols and fatty acids so they are entirely dependent on their hosts for these and other complex lipids. Fatty acid binding proteins (FABPs) of the FABP/P2/CRABP/CRBP family of beta-barrel cytosolic lipid binding proteins (cLBP) appear to be particularly important to schistosomes in the uptake, transport and compartmentalisation of host-derived fatty acids and may provide important targets for immuno- and chemotherapy. Here we describe the isolation of a set of cDNAs prepared from the Asiatic schistosome, Schistosoma japonicum, which encode two groups of cLBPs based on sequence homology and unique cDNA restriction sites. Representative clones from the two groups, one encoding a complete Sj-FABP (F10), and the other encoding a deletion mutant (F25) were characterised at the nucleic acid level by Southern and Northern hybridisation analysis, and at the protein level by immunoblotting. The presence and size of introns in the genes encoding F10 and F25 were determined and, because of the interest in the Schistosoma mansoni FABP homologue (Sm14) as a putative vaccine candidate, the immunogenicity and protective efficacy of the two proteins were also evaluated. A particularly interesting finding was the degree of Sj-FABP amino acid sequence polymorphism found to occur within the S. japonicum worm population, which appears to be greater than that described from cLBPs from vertebrates or, indeed, any other group of organisms investigated to date.

A new member of the transmembrane 4 superfamily (TM4SF) of proteins from schistosomes, expressed by larval and adult Schistosoma japonicum.

The transmembrane 4 superfamily (TM4SF) comprises an assemblage of surface antigens from mammalian cells and from the human blood flukes. Member proteins of the TM4SF are characterized by the presence of four hydrophobic domains, which are presumed to be membrane-spanning, and specific conserved motifs. The Sm23 group of TM4SF, which includes Sm23, Sj23, and Sh23 from blood flukes, shows potential as immunodiagnostic and vaccine target antigens for use in controlling human schistosomiasis. Here we describe a cDNA from miracidia and adult Schistosoma japonicum parasites which apparently encodes a new member of the TM4SF. The deduced polypeptide, termed Sj25/TM4, has substantial amino acid homology to Sm23 from Schistosoma mansoni although it is not a species homologue of Sm23. Sj25/TM4 is predicted to span the cell membrane four times, with its NH2- and COOH-termini embedded in the cytoplasm, and to have two extracellular hydrophilic loops, one of which may be N-glycosylated. This topology is characteristic of TM4SF proteins; in addition, Sj25/TM4 contains the sequence motifs conserved in the TM4SF. Southern hybridization analysis demonstrated that Sj25/TM4 and Sj23 are encoded by genes at separate loci and, further, showed interstrain variation at the locus encoding Sj25/TM4 in Chinese and Philippine isolates of S. japonicum.

Characterisation of a family of Schistosoma japonicum proteins related to dynein light chains.

Dynein light chains (DLC) are components of dynein, an enzyme complex involved in various aspects of microtubule-based motility. We report here the molecular cloning and sequencing of cDNAs encoding a family of DLC-like polypeptides (SjcDLC1-5) from the human bloodfluke Schistosoma japonicum with open reading frames of 87-104 amino acids and deduced molecular masses ranging from 10.5 to 12.3 kDa. Two-dimensional Western blot analysis confirmed the presence of several S. japonicum DLC isoforms with differing pI values and molecular sizes. We also describe the molecular characterisation, genomic organisation and expression of clone SjcDLC1, and the immunological characterisation and localisation of its encoded protein. Northern blot analysis of adult worm RNA indicated SjcDLC1 is encoded by a single message of approximately 650 bp and Southern analysis suggested one SjcDLC1 gene exists in the S. japonicum genome. Immunolocalisation studies demonstrated that the SjcDLC1 protein is present in the tegument of the adult and cercarial stages of S. japonicum. SjcDLC1 and the other SjcDLC may function in the transport of specialised organelles, comprising membranous and discoid bodies, through the tegument to the schistosome-unique heptalaminate tegumental membrane at the external surface of the adult worm. As a consequence, they may provide novel targets for anti-schistosome vaccine and/or drug development.

Brain metastasis of alveolar echinococcosis in a hyperendemic focus of Echinococcus multilocularis infection.

An unusual female case, with alveolar echinococcosis (AE) disseminated from the primary hepatic lesion to the brain by metastasis formation, was retrospectively identified during a community survey in Ningxia Hui Autonomous Region, northwest China in 2003. Among possible metastases of hepatic AE, locations to the brain are rare and usually fatal; and they have especially been assigned to concomitant immune suppression. An enhancing role of pregnancy, which may be suspected in this case, the favourable outcome after surgery and chemotherapy, and also a mental disability in a child following long-term intrauterine exposure to mebendazole, make the report particularly unique.

Gene cloning, overproduction and purification of a functionally active cytoplasmic fatty acid-binding protein (Sj-FABPC) from the human blood fluke Schistosoma japonicum.

We report the gene cloning, molecular characterisation and purification of a 14.7-kDa functionally active recombinant (re) cytoplasmic fatty acid-binding protein (Sj-FABPC) from the Chinese strain of the human bloodfluke Schistosoma japonicum (Sj). As schistosomes are unable to synthesise long chain fatty acids and sterols de novo and must, therefore, take up these lipids from the host, Sj-FABPC is an attractive vaccine and/or drug target. Clone 39 (C39), which contains the entire Sj-FABPC gene, was isolated from a Sj lambda ZAPII cDNA expression library immunoscreened with hyperimmune rabbit serum (HRS) raised against soluble adult Sj proteins. The complete ORF (open reading frame) of Sj-FABPC encodes a protein of 132 amino acids (aa) of 14.7 kDa. The aa sequence of Sj-FABPC exhibits 91% identity to a FABP of S. mansoni (Sm14) and 45% identity to a FABP of Fasciola hepatica (Fh15), putative vaccine candidates for schistosomiasis. Sj-FABPC was subcloned into the QIAexpress vector, pQE-10, and subsequently expressed in Escherichia coli. The re-Sj-FABPC, purified under non-denaturing conditions, was recognized by sera from patients with acute and chronic schistosomiasis japonica. The purified re-Sj-FABPC was also shown to bind to palmitic acid with high affinity. The functional expression of Sj-FABPC will facilitate studies on re-Sj-FABPC to assess its potential as a drug and/or vaccine candidate.

Gulliver, a long terminal repeat retrotransposon from the genome of the oriental blood fluke Schistosoma japonicum.

We characterized the consensus sequence and structure of a long terminal repeat (LTR) retrotransposon from the genome of the human blood fluke, Schistosoma japonicum, and have named this element, Gulliver. The full length, consensus Gulliver LTR retrotransposon was 4788 bp, and it was flanked at its 5'- and 3'-ends by LTRs of 259 bp. Each LTR included RNA polymerase II promoter sequences, a CAAT signal and a TATA box. Gulliver exhibited features characteristic of a functional LTR retrotransposon including two read through (termination) ORFs encoding retroviral gag and pol proteins of 312 and 1071 amino acid residues, respectively. The gag ORF encoded motifs conserved in nucleic acid binding proteins, while the pol ORF encoded conserved domains of aspartic protease, reverse transcriptase (RT), RNaseH and integrase, in that order, a pol pattern conserved in the gypsy lineage of LTR retrotransposons. Whereas the sequence and structure of Gulliver was similar to that of gypsy, phylogenetic analysis revealed that Gulliver did not group particularly closely with the gypsy family. Rather, its closest relatives were a LTR retrotransposon from Caenorhabditis elegans, mag from Bombyx mori and, to a lesser extent, easel from the salmon Oncorhynchus keta. Dot blot hybridizations indicated that Gulliver was present at between 100 and several thousand copies in the S. japonicum genome, and Southern hybridization analysis suggested its probable presence in the genome of Schistosoma mansoni. Transcripts encoding the RT domain of Gulliver were detected by RT-PCR in larval and adult stages of S. japonicum, indicating that (at least) the RT domain of Gulliver is transcribed. This is the first report of the sequence and structure of an LTR retrotransposon from any schistosome or indeed from any species belonging to the phylum Platyhelminthes.

Purification of a recombinant Schistosoma japonicum antigen homologous to the 22-kDa membrane-associated antigen of S. mansoni, a putative vaccine candidate against schistosomiasis.

We describe the cDNA cloning, overproduction and purification of a 22.6-kDa antigen from the human blood fluke Schistosoma japonicum. A 777-bp cDNA (C32) was isolated from a S. japonicum lambda ZAPII cDNA expression library immuno-screened with hyperimmune rabbit serum (HRS) raised against soluble adult S. japonicum proteins. The open reading frame of C32 encodes a protein of 191 amino acids (aa) which exhibits 71% identity to a 22.6-kDa membrane-associated antigen of S. mansoni, a putative vaccine candidate for schistosomiasis. We have identified a sequence motif known as an EF-hand calcium-binding domain in both the S. japonicum and S. mansoni aa sequences, suggesting that the 22.6-kDa antigens are able to bind Ca2+. Further, we have, for the first time, obtained the 22.6-kDa antigen in purified, non-denatured, recombinant form, and in sufficient quantity to assess the protective value of the molecule in vaccination/challenge experiments. This was achieved by synthesizing the schistosome antigen with a short polyhistidine tag fused to the N-terminus which was then used for subsequent affinity purification. The recombinant protein was purified under non-denaturing conditions using nickel-chelate affinity chromatography.