Differential Expression of Hox and Notch Genes in Larval and Adult Stages of Echinococcus granulosus

This investigation aimed to evaluate the differential expression of HoxB7 and notch genes in different developmental stages of Echinococcus granulosus sensu stricto. The expression of HoxB7 gene was observed at all developmental stages. Nevertheless, significant fold differences in the expression level was documented in the juvenile worm with 3 or more proglottids, the germinal layer from infected sheep, and the adult worm from an experimentally infected dog. The notch gene was expressed at all developmental stages of E. granulosus; however, the fold difference was significantly increased at the microcysts in monophasic culture medium and the germinal layer of infected sheep in comparison with other stages. The findings demonstrated that the 2 aforementioned genes evaluated in the present study were differentially expressed at different developmental stages of the parasite and may contribute to some important biological processes of E. granulosus.     

Echinococcus granulosus: evaluation of purified antigens immunoreactivity

A new method is presented for the isolation of purified Echinococcus granulosus antigens from sheep hydatid fluid. Echinococcus granulosus antigens were separated from the host's serum contaminants by absorbing sheep serum proteins with specific immunoabsorbents.No net sensitivity gain was obtained by using these purified antigens rather than crude sheep hydatid fluid in hemagglutination and immunoelectrophoretic tests.The presence of two major antigens (≥ 400,000 and 150,000 MW) was confirmed, the larger component being clearly the most immunoreactive.Evidence of a third slightly antigenic fraction of low molecular weight is presented.The effectiveness of labeled major Echinococcus granulosus antigens in radioimmunoprecipitation test is reported.     

Echinococcus granulosus: ovicidal activity of praziquantel and bunamidine hydrochloride

Eggs of Echinococcus granulosus either released from or contained within the proglottids were treated with praziquantel and bunamidine hydrochloride at 37 C for 1 hr and then administered orally to mice and gerbils to test their infectivity. Praziquantel did not possess absolute ovicidal activity against E. granulosus eggs either within or outside the proglottids and bunamidine hydrochloride did not kill them within the proglottids. These findings bear on the use of praziquantel in control programs.     

Sequence Analysis of cytb Gene in Echinococcus granulosus from Western China

Echinococcus granulosus is the causative agent of cystic echinococcosis with medical and veterinary importance in China. Our main objective was to discuss the genotypes and genetic diversity of E. granulosus present in domestic animals and humans in western China. A total of 45 hydatid cyst samples were collected from sheep, humans, and a yak and subjected to an analysis of the sequences of mitochondrial cytochrome b (cytb) gene. The amplified PCR product for all samples was a 1,068 bp band. The phylogenetic analysis showed that all 45 samples were identified as E. granulosus (genotype G1). Ten haplotypes were detected among the samples, with the main haplotype being H1. The haplotype diversity was 0.626, while the nucleotide diversity was 0.001. These results suggested that genetic diversity was low among our samples collected from the west of China based on cytb gene analysis. These findings may provide more information on molecular characteristics of E. granulosus from this Chinese region.     

A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.     

A recombinant antigen with potential for serodiagnosis of Echinococcus granulosus infection in dogs

Antibodies specific for Echinococcus granulosus were affinity purified from dog serum on transfer blots containing putative serodiagnostic antigens. These antibodies and serum pools derived from dogs with E. granulosus infections were used to screen a lambda gt11 cDNA library constructed using E. granulosus protoscolex mRNA. Nine definitive antigenic clones were isolated and characterized, of which one (c10P1) gave strong specific reactions in plaque immunoassay with sera from E. granulosus infected dogs. These clones were all subcloned into the plasmid vector pGEX-1. Antigenicity of clones was confirmed in colony immunoassay and/or immunoblot. Glutathione S-transferase (GST) fusion proteins of individual subclones were produced in Escherichia coli, purified by affinity chromatography and evaluated in ELISA using sera from dogs with infections of E. granulosus, Taenia spp. or nematodes, and helminth-free dog sera. The GST fusion protein 10P1 showed a specificity of 100% in ELISA for diagnosis of E. granulosus infection in dogs despite its relatively low sensitivity. Further investigations aim to identify additional recombinant antigens and test 10P1 expressed in alternative expression systems to increase diagnostic sensitivity of the ELISA.     

Experimental Evolution of a Trypanosome Parasite of Bumblebees and its Implications for Infection Success and Host Immune Response

Selection on basic growth properties of parasites may have many consequences for parasite traits, infection outcome, or host responses to infection. It is known that genotypes (strains) of the trypanosome parasite of bumblebees Crithidia bombi vary widely in their growth rates in their natural host, Bombus terrestris, as well as when cultured in medium. To test for changes in growth rates and their consequences, we here experimentally evolved six strains of C. bombi for fast and slow growth under controlled conditions in culture medium. Subsequently, we infected the evolved lines in live host and found that lines selected for slow growth attained higher infection intensity in the live bumblebee than those evolved for fast growth, whilst the immune response of the host was the same to both kinds of lines. These results fit the expectation that attenuation through rapid adaptation to a different environment, the culture medium, makes the parasite less successful in its next host. Selection for fast growth therefore does not necessarily lead to higher parasite success or more transmission. Hence, insect trypanosome pathogens can be attenuated by experimental evolution in the culture; this could inform important aspects of host-parasite evolution and perhaps vaccine development.     

Echinococcus granulosus antigen B structure: subunit composition and oligomeric states

Background

Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states.

Methodology/Principal Findings

The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability.

Conclusions/Significance

For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.     

Echinococcus granulosus: cellular territories and morphological regions in mature protoscoleces

Basic aspects of the generation, structure and function of Echinococcus granulosus protoscoleces are unknown. We review the work done on the structure and ultrastructure of the E. granulosus protoscolex and provide new data together with a comprehensive view of this form of the parasite. The surface, as observed by scanning electron microscopy, tightly correlates with five cellular territories characterized in the interior using light and transmission electron microscopy as well as a histochemical technique. Three of these territories are surrounded by a basal lamina that is also present in the internal side of the tegument, suggesting a complex internal organization. These cellular territories correlate with the expression of specific genes and the regionalization of DNA synthesis in protoscoleces. Additionally, a proposal to explain movements of the body of this form of the parasite in relation to the neck or to the germinal layer of the hydatid cyst is provided.     

Echinococcus granulosus: antigen characterization by chemical treatment and enzymatic deglycosylation.

Parasite antigenic fractions obtained by biochemical purification of sheep hydatid fluid were subjected to enzymatic digestion. The relative mobilities of the 5 and B antigens, before and after treatment, were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Antigenic fractions transferred to nitrocellulose were also treated with sodium metaperiodate and concanavalin A. The results indicate that antigen 5 contains a substantial amount of carbohydrates covalently linked to a polypeptide backbone, which strongly bind to concanavalin A and is removed by N-glycosidase F (PNGase F). Antigen 5 possesses complex N-linked oligosaccharides (PNGase F sensitive), without terminal N-acetyl-D-glucosamine residues (N-acetyl-D-glucosaminidase nonsensitive) and has no high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H nonsensitive). In contrast, the antigen B of low molecular weight is not susceptible to either enzymatic digestions (PNGase F, Endo H, and N-acetyl-D-glucosaminidase) or sodium metaperiodate oxidation and it does not bind to concanavalin A. Polyclonal antibodies prepared against the two antigens reacted with the deglycosylated antigen 5 in Western blot. The dominant epitopes are, therefore, polypeptides, although the presence of carbohydrate epitopes in the native glycoproteins cannot be excluded.     

Postcolonial Ecologies of Parasite and Host: Making Parasitism Cosmopolitan

The interest of F. Macfarlane Burnet in host–parasite interactions grew through the 1920s and 1930s, culminating in his book, Biological Aspects of Infectious Disease (1940), often regarded as the founding text of disease ecology. Our knowledge of the influences on Burnet’s ecological thinking is still incomplete. Burnet later attributed much of his conceptual development to his reading of British theoretical biology, especially the work of Julian Huxley and Charles Elton, and regretted he did not study Theobald Smith’s Parasitism and Disease (1934) until after he had formulated his ideas. Scholars also have adduced Burnet’s fascination with natural history and the clinical and public health demands on his research effort, among other influences. I want to consider here additional contributions to Burnet’s ecological thinking, focusing on his intellectual milieu, placing his research in a settler society with exceptional expertise in environmental studies and pest management. In part, an ‘‘ecological turn’’ in Australian science in the 1930s, derived to a degree from British colonial scientific investments, shaped Burnet’s conceptual development. This raises the question of whether we might characterize, in postcolonial fashion, disease ecology, and other studies of parasitism, as successful settler colonial or dominion science.     

Echinococcus granulosus: comparison between antigens in scolices and hydatid fluid.

The sheep hydatid fluid and scolex antigens of Echinococcus granulosus were precipitated by increasing ammonium sulphate concentrations. The antigenic profiles, obtained by complement fixation and indirect hemagglutination inhibition tests on the ammonium sulphate precipitates after linear sucrose gradient ultracentrifugation, were different comparing the hydatid fluid and the scolex extracts. Antigenic non-identity was found between sheep hydatid fluid and scolex extracts by immunodiffusion and indirect hemagglutination inhibition tests. The ammonium sulphate precipitates of hydatid fluid and scolex extracts revealed several different bands by slab-gel examination.