Polycystic echinococcosis (PE) is a zoonosis endemic in the Neotropical region of the Americas. It is caused by the larval stage of the cestode Echinococcus vogeli, which develops as harmful cysts that slowly grow in the liver, lungs and other organs of humans and other host species. Human PE diagnosis is usually based on clinical and epidemiological aspects and imaging techniques, often requiring confirmation by immunological assays. The currently available serological tests for detecting antibodies against Echinococcus spp. are mostly based on complex, variable and poorly characterized mixtures of native parasite antigens, which impairs specificity and/or sensitivity. In this scenario, the evaluation of well-characterized alternative antigens is urgently needed for the improvement of PE diagnosis. Here, two subunits (AgB8/1 and AgB8/2) of the major secretory antigen from Echinococcus granulosus (antigen B (AgB)), of diagnostic value for cystic echinococcosis, were validated for PE diagnosis. These antigens, produced as pure recombinant proteins (rAgB8/1 and rAgB8/2) in Escherichia coli, allowed detecting specific immunoglobulin G antibodies in sera from PE patients in an enzyme-linked immunosorbent assay, with sensitivities of 83.72% and 81.40%, respectively, and specificities of 83.12% and 80.09%, respectively. The use of recombinant proteins overcomes difficulties to obtain parasite material and reduced non-specific reactions and costs. Our results demonstrated reproducibility and accuracy high enough to be considered valid according to the acceptance criteria for Food and Drug Administration assay validation. This qualifies rAgB8/1 and rAgB8/2 as potential substitutes for the currently used parasite crude or partially purified antigens.

Totipotent somatic stem cells (neoblasts) are key players in the biology of flatworms and account for their amazing regenerative capability and developmental plasticity. During recent years, considerable progress has been made in elucidating molecular features of neoblasts from free-living flatworms, whereas their role in parasitic species has so far merely been addressed by descriptive studies. Very recently, however, significant advances have been made in the in vitro culture of neoblasts from the cestode Echinococcus multilocularis. The isolated cells proved capable of generating mature metacestode vesicles under laboratory conditions in a manner that closely resembles the oncosphere-metacestode transition during natural infections. Using the established neoblast cultivation protocols, combined with targeted manipulation of Echinococcus genes by RNA-interference, several fundamental questions of host-dependent parasite development can now be addressed. Here, I give an overview of current cultivation techniques for E. multilocularis neoblasts and present experimental approaches to study their function. Furthermore, I introduce the E. multilocularis genome sequencing project that is presently in an advanced stage. The combined input of data from the E. multilocularis sequencing project, stem cell cultivation, and recently initiated attempts to genetically manipulate Echinococcus will provide an ideal platform for hypothesis-driven research into cestode development in the next years.

Four monoclonal antibodies (MAb) to Echinococcus granulosus Antigen 5 (Ag5) and Antigen B (AgB) were assessed in an enzyme-linked immunosorbent assay (ELISA) for detection of circulating antigens (CAg) in sera of human patients with E. granulosus infection. Around 5·5–8% of 200 sera from 42 surgically proven hydatid patients contained detectable CAg by individual MAb. The combined detection rate for CAg, using four MAb, was 19% (38/200). Although hydatid CAg was detected by MAb in at least one serum sample from 21 of 42 patients, some patients remained negative in the assay regardless of the time when serum samples were taken (pre- or post-operatively), or of the continuing presence of hydatid cysts, their location or fertility. In addition, it was observed that the binding capacity of MAb for sheep hydatid cyst fluid antigen (SHCF) was somewhat reduced in the presence of normal human serum. The CAg detection assay would only be useful for assessment of hydatid infection status in patients with detectable CAg in serum samples.

An antibody competition enzyme-linked immunosorbent assay (ELISA) using 4 different monoclonal antibodies (MAb) raised against major antigens (Antigen 5 and Antigen B) of Echinococcus granulosus was evaluated for the diagnosis of human hydatidosis. The competition assay, using anti-Ag5 MAb 24.14, detected specific antibodies in 70% (131/188) of sera from patients with surgically confirmed E. granulosus infection and 38·5% (10/26) of sera from patients with E. multilocularis infection. None of the sera from patients with Taenia solium cysticercosis (10), T. saginata (2), filariasis (22), strongyloidiasis (19), fascioliasis (4), bilharziasis (4) and amoebiasis (2) tested were positive using a cut-off point established through reaction between MAb 24.14 and normal human sera. The combined use of the MAb 24.14-based competition ELISA with the conventional antibody-binding assay provides a highly sensitive (92·8%) and specific screening system for human hydatid disease diagnosis.

Numerous genetic variants of the Echinococcus antigen B (AgB) are encountered within a single metacestode. This could be a reflection of gene redundancy or the result of a somatic hypermutation process. We evaluate the complexity of the AgB multigene family by characterizing the upstream promoter regions of the 4 already known genes (EgAgB1-EgAgB4) and evaluating their redundancy in the genome of 3 Echinococcus species (E. granulosus, E. ortleppi and E. multilocularis) using PCR-based approaches. We have ascertained that the number of AgB gene copies is quite variable, both within and between species. The most repetitive gene seems to be AgB3, of which there are more than 110 copies in E. ortleppi. For E. granulosus, we have cloned and characterized 10 distinct upstream promoter regions of AgB3 from a single metacestode. Our sequences suggest that AgB1 and AgB3 are involved in gene conversion. These results are discussed in light of the role of gene redundancy and recombination in parasite evasion mechanisms of host immunity, which at present are known for protozoan organisms, but virtually unknown for multicellular parasites.

Echinococcus granulosus antigen B (AgB) is encoded by a gene family and is involved in the evasion of the host immune response. E. granulosus exists as a number of strains (G1–G10) that differ in biological characteristics. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and transcription profile of AgB in 5 E. granulosus strainsNucleotide sequence data reported in this paper are available in the GenBankTM database under the Accession numbers AY569341-AY569371, AY159484, AY159485, AY608606, AY608607, AY614001.. Twenty-four genomic sequences were isolated and clustered in 3 groups related to 2 of the 5 reported AgB genes. AgB4 genes were present in almost all strains, whereas AgB2 were present as functional genes exclusively in G1/G2 cluster, and as non-functional genes in G5 and the G6/G7 cluster, suggesting inter-strain variation. The AgB transcription patterns, analysed by RT-PCR, showed that AgB2 and AgB4 genes were transcribed in G1, while only the AgB4 gene was transcribed in G7 strain. Cysts from the same strain or cluster shared more genomic and cDNA variants than cysts from different strain or cluster. The level of nucleotide and deduced amino acid sequence variation observed is higher than that reported so far for coding genes of other helminths. Neutrality was rejected for AgB2 genes. These data show the genetic polymorphism of antigen-coding genes among genetically characterized strains of E. granulosus.