Comparative study on secretome and transmembranome of immature and mature metacestodes of Echinococcus multilocularis.

Alveolar echinococcosis (AE) is a worldwide zoonosis caused by E. multilocularis. Humans become infected through oral ingestion of the eggs. Host of E. multilocularis produces immune responses that help to either reject and/or limit the growth of this parasite, and in response the parasite produces molecules against this immune attack. This study identifies candidate key molecules in the early infection phase and the chronic stage of the parasite infestation, through comparison of gene expression of 4- and 16-week metacestodes. First, RNA was isolated from 4- and 16-weeks metacestodes of E. multilocularis (Nemuro strain). Thereafter, clean reads with lengths of 50bp or longer were compared against a reference genome using TopHat. Functional annotation of transcripts of E. multilocularis were investigated using multi-step bioinformatics tools. At the gene ontology (GO) level, 356 and 1774 transmembrane (TM) predicted proteins of the E. multilocularis were mapped to an enhanced 'hydrolase activity' and increased 'transmembrane transporter activity', respectively. In addition, comparison of gene expression level between 4- and 16-week metacestode revealed 168 different expression (DE) genes. This study has demonstrated that, the expression levels of predicted ES and TM proteins in E. multilocularis change in the transformation from one stage to another. Genes that are highly expressed in immature or mature metacestode could be explored as novel candidates for diagnostic antigens and vaccine targets.

Analysis on Gene Expression Profile in Oncospheres and Early Stage Metacestodes from Echinococcus multilocularis.

Alveolar echinococcosis is a worldwide zoonosis of great public health concern. Analysis of genome data for Echinococcus multilocularis has identified antigen families that can be used in diagnostic assays and vaccine development. However, little gene expression data is available for antigens of the egg and early larval stages. To address this information gap, we used a Next-Generation Sequencing approach to investigate three different stages (non-activated and activated oncospheres, and early stage metacestodes) of E. multilocularis (Nemuro strain). Transcriptome data analysis revealed that some diagnostic antigen gp50 isoforms and the antigen Eg95 family dominated in activated oncospheres, and the antigen B family dominated in early stage metacestodes. Furthermore, heat shock proteins and antigen II/3 are constantly expressed in the three stages. The expression pattern of various known antigens in E. multilocularis may give fundamental information for choosing candidate genes used in diagnosis and vaccine development.

Where to deliver baits for deworming urban red foxes for Echinococcus multilocularis control: new protocol for micro-habitat modeling of fox denning requirements.

BACKGROUND: Deworming wild foxes by baiting with the anthelmintic praziquantel is being established as a preventive technique against environmental contamination with Echinococcus multilocularis eggs. Improvement of the cost-benefit performance of baiting treatment is required urgently to raise and maintain the efficacy of deworming. We established a spatial model of den site selection by urban red foxes, the definitive host, to specify the optimal micro-habitats for delivering baits in a new modeling approach modified for urban fox populations. METHODS: The model was established for two cities (Obihiro and Sapporo) in Hokkaido, Japan, in which a sylvatic cycle of E. multilocularis is maintained. The two cities have different degrees of urbanization. The modeling process was designed to detect the best combination of key environmental factors and spatial scale that foxes pay attention to most (here named 'heeding range') when they select den sites. All possible models were generated using logistic regression analysis, with "presence" or "absence" of fox den as the objective variable, and nine landscape categories customized for urban environments as predictor variables to detect the best subset of predictors. This procedure was conducted for each of ten sizes of concentric circles from dens and control points to detect the best circle size. Out of all models generated, the most parsimonious model was selected using Akaike's Information Criterion (AIC) inspection. RESULTS: Our models suggest that fox dens in Obihiro are located at the center of a circle with 500 m radius including low percentages of wide roads, narrow roads, and occupied buildings, but high percentages of green covered areas; the dens in Sapporo within 300 m radius with low percentages of wide roads, occupied buildings, but high percentages of riverbeds and green covered areas. The variation of the models suggests the necessity of accumulating models for various types of cities in order to reveal the patterns of the model. CONCLUSIONS: Our denning models indicating suitable sites for delivering baits will improve the cost-benefit performance of the campaign. Our modeling protocol is suitable for the urban landscapes, and for extracting the heeding range when they select the den sites.

Characterization of a surface glycoprotein from Echinococcus multilocularis and its mucosal vaccine potential in dogs.

Alveolar echinococcosis is a refractory disease caused by the metacestode stage of Echinococcus multilocularis. The life cycle of this parasite is maintained primarily between foxes and many species of rodents; thus, dogs are thought to be a minor definitive host except in some endemic areas. However, dogs are highly susceptible to E. multilocularis infection. Because of the close contact between dogs and humans, infection of dogs with this parasite can be an important risk to human health. Therefore, new measures and tools to control and prevent parasite transmission required. Using 2-dimensional electrophoresis followed by western blot (2D-WB) analysis, a large glycoprotein component of protoscoleces was identified based on reactivity to intestinal IgA in dogs experimentally infected with E. multilocularis. This component, designated SRf1, was purified by gel filtration using a Superose 6 column. Glycosylation analysis and immunostaining revealed that SRf1 could be distinguished from Em2, a major mucin-type antigen of E. multilocularis. Dogs (n=6) were immunized intranasally with 500 µg of SRf1 with cholera toxin subunit B by using a spray syringe, and a booster was given orally using an enteric capsule containing 15 mg of the same antigen. As a result, dogs immunized with this antigen showed an 87.6% reduction in worm numbers compared to control dogs (n=5) who received only PBS administration. A weak serum antibody response was observed in SRf1-immunized dogs, but there was no correlation between antibody response and worm number. We demonstrated for the first time that mucosal immunization using SRf1, a glycoprotein component newly isolated from E. multilocularis protoscoleces, induced a protection response to E. multilocularis infection in dogs. Thus, our data indicated that mucosal immunization using surface antigens will be an important tool to facilitate the development of practical vaccines for definitive hosts.

Molecular phylogeny of the genus Taenia (Cestoda: Taeniidae): proposals for the resurrection of Hydatigera Lamarck, 1816 and the creation of a new genus Versteria.

The cestode family Taeniidae generally consists of two valid genera, Taenia and Echinococcus. The genus Echinococcus is monophyletic due to a remarkable similarity in morphology, features of development and genetic makeup. By contrast, Taenia is a highly diverse group formerly made up of different genera. Recent molecular phylogenetic analyses strongly suggest the paraphyly of Taenia. To clarify the genetic relationships among the representative members of Taenia, molecular phylogenies were constructed using nuclear and mitochondrial genes. The nuclear phylogenetic trees of 18S ribosomal DNA and concatenated exon regions of protein-coding genes (phosphoenolpyruvate carboxykinase and DNA polymerase delta) demonstrated that both Taenia mustelae and a clade formed by Taenia parva, Taenia krepkogorski and Taenia taeniaeformis are only distantly related to the other members of Taenia. Similar topologies were recovered in mitochondrial genomic analyses using 12 complete protein-coding genes. A sister relationship between T. mustelae and Echinococcus spp. was supported, especially in protein-coding gene trees inferred from both nuclear and mitochondrial data sets. Based on these results, we propose the resurrection of Hydatigera Lamarck, 1816 for T. parva, T. krepkogorski and T. taeniaeformis and the creation of a new genus, Versteria, for T. mustelae. Due to obvious morphological and ecological similarities, Taenia brachyacantha is also included in Versteria gen. nov., although molecular evidence is not available. Taenia taeniaeformis has been historically regarded as a single species but the present data clearly demonstrate that it consists of two cryptic species.

Molecular survey of Tritrichomonas suis (=T. foetus) 'cat' and 'cattle' genotypes in pigs in Japan.

Tritrichomonas suis (=T. foetus) is a protozoan parasite of pigs, cattle and cats. Based on host range and genetic differences, T. suis has been divided into a 'cat genotype' and a 'cattle genotype', with the latter genotype capable of infecting both cattle and pigs. Since no information is currently available on the genetic characteristics of T. suis from pigs in Japan, we conducted a molecular survey of T. suis using fecal DNA from pigs in Japan. Of the 64 pigs examined, nested PCR revealed that 36 (56.3%) were positive for T. suis. Sequence analysis of 8 positive samples showed that 7 of the pig isolates belonged to the 'cattle genotype' and the remaining isolate belonged to the 'cat genotype'. The findings revealed that T. suis infection is common in pigs in Japan and that pigs can be infected by both genotypes.

Mucosal adjuvanticity of fibronectin-binding peptide (FBP) fused with Echinococcus multilocularis tetraspanin 3: systemic and local antibody responses.

BACKGROUND: Studies have shown that a bacterial fibronectin attachment protein (FAP) is able to stimulate strong systemic and mucosal antibody responses when it is used alone or co-administrated with other antigens (Ags). Thus, it has been suggested to be a promising adjuvant candidate for the development of efficient vaccines. However, the co-administered Ags and FAP were cloned, expressed and purified individually to date. In a recent study, we first evaluated the adjuvanticity of a fibronectin-binding peptide (FBP, 24 amino acids) of Mycobacterium avium FAP fused with Echinococcus multilocularis tetraspanin 3 (Em-TSP3) by detecting systemic and local antibody responses in intranasally (i.n.) immunized BALB/c mice. METHODOLOGY/PRINCIPAL FINDINGS: Em-TSP3 and FBP fragments were linked with a GSGGSG linker and expressed as a single fusion protein (Em-TSP3-FBP) using the pBAD/Thio-TOPO expression vector. BALB/c mice were immunized i.n. with recombinant Em-TSP3-FBP (rEm-TSP3-FBP) and rEm-TSP3+CpG and the systemic and local antibody responses were detected by ELISA. The results showed that both rEm-TSP3-FBP and rEm-TSP3+CpG evoked strong serum IgG (p<0.001) and IgG1 responses (p<0.001), whereas only the latter induced a high level IgG2α production (p<0.001), compared to that of rEm-TSP3 alone without any adjuvant. There were no significant differences in IgG and IgG1 production between the groups. Low level of serum IgA and IgM were detected in both groups. The tendency of Th1 and Th2 cell immune responses were assessed via detecting the IgG1/IgG2α ratio after the second and third immunizations. The results indicated that i.n. immunization with rEm-TSP3-FBP resulted in an increased IgG1/IgG2α ratio (a Th2 tendency), while rEm-TSP3+CpG caused a rapid Th1 response that later shifted to a Th2 response. Immunization with rEm-TSP3-FBP provoked significantly stronger IgA antibody responses in intestine (p<0.05), lung (p<0.001) and spleen (p<0.001) compared to those by rEm-TSP3+CpG. Significantly high level IgA antibodies were detected in nasal cavity (p<0.05) and liver (p<0.05) samples from both groups when compared to rEm-TSP3 alone without any adjuvant, with no significant difference between them. CONCLUSIONS: I.n. administration of rEm-TSP3-FBP can induce strong systemic and mucosal antibody responses in immunized BALB/c mice, suggesting that fusion of Em-TSP3 with FBP is a novel, prospective strategy for developing safe and efficient human mucosal vaccines against alveolar echinococcosis (AE).

A pilot study on developing mucosal vaccine against alveolar echinococcosis (AE) using recombinant tetraspanin 3: Vaccine efficacy and immunology.

BACKGROUND: We have previously evaluated the vaccine efficacies of seven tetraspanins of Echinococcus multilocularis (Em-TSP1-7) against alveolar echinococcosis (AE) by subcutaneous (s.c.) administration with Freund's adjuvant. Over 85% of liver cyst lesion number reductions (CLNR) were achieved by recombinant Em-TSP1 (rEm-TSP1) and -TSP3 (rEm-TSP3). However, to develop an efficient and safe human vaccine, the efficacy of TSP mucosal vaccines must be thoroughly evaluated. METHODOLOGY/PRINCIPAL FINDINGS: rEm-TSP1 and -TSP3 along with nontoxic CpG ODN (CpG oligodeoxynucleotides) adjuvant were intranasally (i.n.) immunized to BALB/c mice and their vaccine efficacies were evaluated by counting liver CLNR (experiment I). 37.1% (p < 0.05) and 62.1% (p < 0.001) of CLNR were achieved by these two proteins, respectively. To study the protection-associated immune responses induced by rEm-TSP3 via different immunization routes (i.n. administration with CpG or s.c. immunization with Freund's adjuvant), the systemic and mucosal antibody responses were detected by ELISA (experiment II). S.c. and i.n. administration of rEm-TSP3 achieved 81.9% (p < 0.001) and 62.8% (p < 0.01) CLNR in the liver, respectively. Both the immunization routes evoked strong serum IgG, IgG1 and IgG2α responses; i.n. immunization induced significantly higher IgA responses in nasal cavity and intestine compared with s.c. immunization (p < 0.001). Both immunization routes induced extremely strong liver IgA antibody responses (p < 0.001). The Th1 and Th2 cell responses were assessed by examining the IgG1/IgG2α ratio at two and three weeks post-immunization. S.c. immunization resulted in a reduction in the IgG1/IgG2α ratio (Th1 tendency), whereas i.n. immunization caused a shift from Th1 to Th2. Moreover, immunohistochemistry showed that Em-TSP1 and -TSP3 were extensively located on the surface of E. multilocularis cysts, protoscoleces and adult worms with additional expression of Em-TSP3 in the inner part of protoscoleces and oncospheres. CONCLUSIONS: Our study indicated that i.n. administration of rEm-TSP3 with CpG is able to induce both systemic and local immune responses and thus provides significant protection against AE.

Trichinella spiralis: infection changes serum paraoxonase-1 levels, lipid profile, and oxidative status in rats.

Paraoxonase-1 (PON1) is an HDL-associated enzyme with anti-atherogenic properties. Reduced PON1 activity has previously been observed in Nippostrongylus brasiliensis-infected rats. However, the effect of chronic zoonotic nematode infections on serum PON1 activity has not yet been studied. Therefore, we evaluated the effect of Trichinella spiralis infection on serum PON1 activity, the lipid profile, and oxidative stress in rats. There were significant reductions in serum PON1 activities (Day 2-Week 7 post-infection) in rats infected with T. spiralis, and these reductions were associated with significant increases in the serum levels of triglyceride and LDL/VLDL, as well as a significant reduction in the level of HDL. Moreover, T. spiralis infection was associated with a status of oxidative stress indicated by increased concentrations of superoxide dismutase and malondialdehyde. Given the zoonotic prevalence of T. spiralis and the cardioprotective role of PON1, further mechanistic research in this area is warranted.

Zoophilic feeding behaviour of phlebotomine sand flies in the endemic areas of cutaneous leishmaniasis of Sindh Province, Pakistan.

Leishmania (Leishmania) major has been identified as the major causative agent of cutaneous leishmaniasis in Sindh Province of southern Pakistan. To make a rational approach for understanding the pathogen transmission cycles, the sand fly species and their natural blood meals in the endemic areas were examined. Total DNA was individually extracted from sand flies collected in four villages in Sindh Province. PCR-RFLP (restriction fragment length polymorphism) and sequence analysis of the 18S ribosomal RNA gene revealed that female sand flies identified were Sergentomyia clydei/Sergentomyia ghesquierei/Sergentomyia magna (68.6%), Sergentomyia dubia (17.1%), Phlebotomus papatasi (7.4%), Phlebotomus alexandri-like sand flies (3.4%) and Sergentomyia dentata (3.4%). PCR amplification of leishmanial kinetoplast DNA did not result in positive signals, suggesting that all 175 tested female sand flies were not infected with leishmanial parasites or contained undetectable levels of leishmanial DNA. Amplification and sequencing of the vertebrate cytochrome b gene in 28 blood-fed sand flies revealed that P. papatasi fed on cattle and wild rat whereas P. alexandri-like specimens fed on human, cattle, goat and dog. Although Sergentomyia sand flies are generally known to feed on cold-blooded animals, S. clydei, S. dubia and S. ghesquierei preferred humans, cattle, goat, sheep, buffalo, dog, donkey, wild rat and Indian gerbil. The epidemiological significance of the zoophilic feeding on various host species by Phlebotomus and Sergentomyia sand flies in Pakistan is further required to study for better understanding the zoonotic transmission of sand-fly-borne pathogens and for appropriate management of the vectors.

Intestinal Tritrichomonas suis (=T. foetus) infection in Japanese cats.

Tritrichomonas suis (=T. foetus) has recently been reported to be a causative agent of chronic large-bowel diarrhea in cats. While the disease was previously attributed to Pentatrichomonas hominis, the etiologic agent for feline trichomonal diarrhea was identified as T. suis. Although feline trichomonosis due to T. suis has been reported at prevalences ranging from 14 to 31% in Europe and the U.S., no reports of the pathogen have been published to date in Japan. In 2008, however, we encountered a case of feline trichomonosis at the Veterinary Teaching Hospital of Hokkaido University. The parasite was identified as T. suis by nested PCR amplification of partial internal transcribed spacer region 1 and 5.8S ribosomal RNA gene sequences with T. suis-specific primers and DNA sequencing of the amplified products. We then conducted surveys for feline trichomonosis in three different animal hospitals using either cultivation and/or PCR-based assays. The results revealed that 13 of 147 samples (8.8%) were positive for T. suis, and that 5 of the 13 infected cats, which ranged between 1 month and 7.5 years-old, showed chronic diarrhea. Seven of the infected cats were purebred and 6 were mixed breed. These findings suggested that feline trichomonosis is prevalent in Japan, and that T. suis may play a role as a causative agent of feline chronic diarrhea.

Identification of genetic loci affecting the establishment and development of Echinococcus multilocularis larvae in mice.

Alveolar echinococcosis (AE) is a severe hepatic disorder caused by larval infection by the fox tapeworm Echinococcus multilocularis. The course of parasitic development and host reactions are known to vary significantly among host species, and even among different inbred strains of mice. As reported previously, after oral administration of parasite eggs, DBA/2 (D2) mice showed a higher rate of cyst establishment and more advanced protoscolex development in the liver than C57BL/6 (B6) mice. These findings strongly suggest that the outcome of AE is affected by host genetic factor(s). In the present study, the genetic basis of such strain-specific differences in susceptibility/resistance to AE in murine models was studied by whole-genome scanning for quantitative trait loci (QTLs) using a backcross of (B6×D2)F(1) and D2 mice with varying susceptibility to E. multilocularis infection. For cyst establishment, genome linkage analysis identified one suggestive and one significant QTL on chromosomes (Chrs.) 9 and 6, respectively, whereas for protoscolex development, two suggestive and one highly significant QTLs were detected on Chrs. 6, 17 and 1, respectively. Our QTL analyses using murine AE models revealed that multiple genetic factors regulated host susceptibility/resistance to E. multilocularis infection. Moreover, our findings show that establishment of the parasite cysts in the liver is affected by QTLs that are distinct from those associated with the subsequent protoscolex development of the parasite, indicating that different host factors are involved in the host-parasite interplay at each developmental stage of the larval parasite. Further identification of responsible genes located on the identified QTLs could lead to the development of effective disease prevention and control strategies, including an intensive screening and clinical follow-up of genetically high-risk groups for AE infection.

Echinococcus multilocularis: purification and characterization of glycoprotein antigens with serodiagnostic potential for canine infection.

We show that a conventionally purified glycoprotein component of Echinococcus multilocularis protoscolex, designated as Emgp-89, may be useful as a serodiagnostic antigen for detecting E. multilocularis infection in dogs domesticated in endemic areas. Emgp-89 was obtained from the parasite material by a simple procedure using Con A-agarose and subsequent gel filtration chromatography. The purified fraction showed a molecular weight of >4000kDa upon gel filtration and reacted with a series of lectins that specifically bind to mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Subsequently, serodiagnostic performance of Emgp-89 was evaluated through enzyme-linked immunosorbent assays (ELISAs) by using sera from normal, domestic dogs and dogs infected with other helminths. Emgp-89 positively reacted with all 16 serum samples from E. multilocularis-infected dogs, thus showing that this antigen is highly sensitive. On the other hand, the specificity of Emgp-89-based ELISA, determined using 41 serum samples from dogs infected with other helminths, was relatively low (83%). As an attempt to improve the specificity of Emgp-89-based ELISA, we pretreated Emgp-89 with proteinase K or sodium periodate, expecting that these treatments would enable discrimination of true positives from false positives. The ELISA value increased after treatment with sodium periodate in most false-positive samples, whereas significant decreases were observed in sera from all dogs infected with E. multilocularis. Further evaluation of this antigen should be performed using sera from dogs infected with closely-related parasites, including taeniid cestodes, which are expected to prove that this serodiagnostic system is sufficiently specific for clinical and field applications.

Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids.

We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples.

Gene silencing in Echinococcus multilocularis protoscoleces using RNA interference.

We investigated the potential of gene silencing in Echinococcus multilocularis protoscoleces using RNA interference (RNAi). For the introduction of siRNA, soaking and electroporation were first examined for their effects on the viability of protoscoleces and their efficacy for siRNA introduction. Consequently, electroporation using 100 V and 800 µF showed the optimal results. This electroporation procedure was then evaluated for its ability to induce RNAi in protoscoleces using siRNAs targeting the 14-3-3 and elp genes. It was found that the levels of 14-3-3 and elp mRNA in 14-3-3 siRNA- and elp siRNA-treated protoscoleces were reduced to 21.8 ± 2.6 and 35.5 ± 0.4% of those of the untreated control by day 3, respectively. Moreover, the target proteins significantly decreased in the siRNA-treated samples by day 15. In the analysis of viability, the untreated control, electroporation control, 14-3-3 siRNA-treated, and elp siRNA-treated samples displayed 98.4 ± 1.4, 83.0 ± 2.5, 58.0 ± 23.0, and 55.1 ± 14.6% viability, respectively, on day 15. In conclusion, we successfully demonstrated that RNAi mediated the knock-down of target gene expression in E. multilocularis protoscoleces at both the transcriptional and translational levels.

Primary alveolar echinococcosis: course of larval development and antibody responses in intermediate host rodents with different genetic backgrounds after oral infection with eggs of Echinococcus multilocularis.

We investigated parasite establishment, subsequent larval development and antibody responses in gerbils, cotton rats and 4 inbred mouse strains until 16 weeks post inoculation (p.i.) with 200 eggs of Echinococcus multilocularis. The rate of parasite establishment in the liver determined at 4 weeks p.i. was highest in DBA/2, followed by AKR/N, C57BL/10 and C57BL/6 mice, whereas gerbils harboured few parasite foci. The accurate number of liver lesions in cotton rats could not be determined due to rapid growth and advanced multivesiculation of the parasite observed at 2 weeks p.i. The course of larval development was most advanced in DBA/2 mice with mature protoscolex formation at 16 weeks p.i., followed by AKR/N harbouring metacestodes with sparsely distributed immature protoscoleces. On the other hand, C57BL/6 and C57BL/10 mice had infertile metacestodes without any protoscolex formation. The parasite growth in mice was totally slower than those in gerbils and cotton rats. Specific IgG and IgM responses against 3 types of native crude antigens of larval E. multilocularis were evaluated using somatic extracts of and vesicle fluid of metacestode, and somatic extracts from purified protoscoleces. The 4 mouse strains demonstrated basically similar kinetics with apparent IgG and IgM increases at 9 weeks p.i. and thereafter, except C57BL/10, exhibited higher levels of IgM against crude antigens at some time point of infection. On the other hand, a follow-up determination of specific IgG and IgM levels against recombinant antigens from larval E. multilocularis revealed that each mouse strain showed different antibody-level kinetics. The findings in the present study demonstrate that the course of host-parasite interactions in primary alveolar echinococcosis, caused by larval E. multilocularis, clearly varies among intermediate host rodents with different genetic backgrounds.

Echinococcus multilocularis primary cells: improved isolation, small-scale cultivation and RNA interference.

In this study we demonstrate RNA interference mediated knock-down of target gene expression in Echinococcus multilocularis primary cells on both the transcriptional and translational level. In addition, we report on an improved method for generating E. multilocularis primary cell mini-aggregates from in vitro cultivated metacestode vesicles, and on the cultivation of small numbers of small interfering RNA-transfected cells in vitro over an extended period of time. This allows assessments on the effects of RNA interference performed on Echinococcus primary cells with regard to growth, proliferation, differentiation of the parasite and the formation of novel metacestode vesicles in vitro.

Molecular phylogeny of the subfamily Gerbillinae (Muridae, Rodentia) with emphasis on species living in the Xinjiang-Uygur Autonomous Region of China and based on the mitochondrial cytochrome b and cytochrome c oxidase subunit II genes.

Rodents belonging to the subfamily Gerbillinae and living in the Xinjiang-Uygur autonomous region of China were collected in field surveys between 2001 and 2003. We found four Meriones species, including M. chengi M. liycus, M. meridianus, and M. tamariscinus, as well as related species from different genera, Rhombomys opimus and Brachiones przewaliskii For phylogenetic analyses of these gerbilline species, DNA sequences of parts of the mitochondrial cytochrome b (Cytb) and cytochrome c oxidase subunit II (COII) genes were examined with the neighbor Joining, maximum parsimony, maximum likelihood, and Bayesian inference methods. Our phylogenetic analyses suggest that the genus Meriones is not monophyletic and place M. tamaricinus as the sister taxon to a clade comprising Brachiones, Psammomys, Rhombomys, and the other Meriones species. The remaining Meriones species separate into three lineages: M. meridianus (including M. chengi), Meriones unguiculatus, and a clade that includes multiple Meriones species originating from Asia, the Middle East, and Africa. The phylogenetic relationships among the genera Brachines, Meriones, Psammomys, and Rhombomys remain ambiguous, probably due to the saturation of mutations that occurs in fast-evolving mitochondrial DNA. In addition, intraspecific variation was observed for M. meridianus, and this mostly correlated with collection localities, i.e., the northern and southern parts of the Xinjiang region. This variation corresponded to interspecific levels of divergence among other lineages of Meriones. Interestingly, no differences were observed in either the Cytb or COII gene sequences isolated from M. chengi collected from the Turfan Basin in the north and those from M. meridianus in the south, suggesting that M. chengi may be a synonym of M. meridianus.

Echinococcus multilocularis: two-dimensional Western blotting method for the identification and expression analysis of immunogenic proteins in infected dogs.

Domesticated dogs are an important potential source of Echinococcus multilocularis infection in humans; therefore, new molecular approaches for the prevention of the parasite infection in dogs need to be developed. Here, we identified and characterized an immunogenic protein of the parasite by using a proteome-based approach. The total protein extracted from protoscoleces was subjected to two-dimensional Western blotting with sera from dogs experimentally infected with E. multilocularis. Two protein spots showed major reactivity to the sera from infected dogs. The N-terminal amino acid sequences of these spots were identical to the deduced amino acid sequence of the product of the putative hsp20 gene. RT-PCR and Western blot analyses revealed that the putative hsp20 gene and its products were expressed in almost all stages of the parasite life cycle. Furthermore, recombinant hsp20 showed specific reactivity to the sera from infected dogs, suggesting that this molecule may facilitate the development of a practical vaccine.

Evaluation of Myanmar medicinal plant extracts for antitrypanosomal and cytotoxic activities.

Current chemotherapeutic options for African trypanosomiasis in humans and livestock are very limited. In the present study, a total of 71 medicinal plant specimens from 60 plant species collected in Myanmar were screened for antitrypanosomal activity against trypomastigotes of Trypanosoma evansi and cytotoxicity against MRC-5 cells in vitro. The methanol extract of dried rootbark of Vitis repens showed the highest antitrypanosomal activity with IC(50) value of 8.6 +/- 1.5 microg/ml and the highest selectivity index of 24.4. The extracts of Brucea javanica, Vitex arborea, Eucalyptus globulus and Jatropha podagrica had also remarkable activity with IC(50) values and selectivity indices in the range of 27.2-52.6 microg/ml and 11.4-15.1 respectively.