Lipid-free Antigen B subunits from echinococcus granulosus: oligomerization, ligand binding, and membrane interaction properties

Background: The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied.<p></p> Methodology/Principal Findings: Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles.<p></p> Conclusions/Significance: We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy.<p></p&gt

Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations

A sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Echinococcus multilocularis coproantigens (EM-ELISA) was developed with polyclonal rabbit (solid phase) and chicken egg (catching) antibodies that were directed against E. multilocularis coproantigens and somatic worm antigens, respectively. In experimentally infected dogs and cats, coproantigens were first detectable 6-17 days postinfection (PI) in samples of 8 dogs (worm burdens at necropsy: 6,330-43,200) and from 11 days PI onward in samples of 5 cats infected with 20-6,833 worms. After anthelmintic treatment of 4 dogs and 5 cats at day 20 PI, coproantigen excretion disappeared within 3-5 days. The sensitivity of the ELISA was 83.6% in 55 foxes infected with 4-60,000 E. multilocularis, but reached 93.3% in the 45 foxes harboring more than 20 worms. The EM-ELISA was used in surveys of 'normal' dog and cat populations in Switzerland. Among 660 dogs and 263 cats, 5 dogs and 2 cats exhibited a positive reaction. In 2 of these dogs (0.30%) and 1 cat (0.38%), intestinal E. multilocularis infections were confirmed by necropsy, polymerase chain reaction PCR, or both. The specificities of the ELISA in these groups were found to be 99.5% and 99.6%, respectively, if positive ELISA results that could not be confirmed by other methods were classified as 'false positive' reactions

In Vitro and In Vivo Effects of Tamoxifen against Larval Stage Echinococcus granulosus

Cystic echinococcosis is a zoonotic infection caused by the larval stage of the cestode Echinococcus granulosus. Chemotherapy currently employs benzimidazoles; however, 40% of cases do not respond favorably. With regard to these difficulties, novel therapeutic tools are needed to optimize treatment in humans. The aim of this work was to explore the in vitro and in vivo effects of tamoxifen (TAM) against E. granulosus. In addition, possible mechanisms for the susceptibility of TAM are discussed in relation to calcium homeostasis, P-glycoprotein inhibition, and antagonist effects on a putative steroid receptor. After 24 h of treatment, TAM, at a low micromolar concentration range (10 to 50 μM), inhibited the survival of E. granulosus protoscoleces and metacestodes. Moreover, we demonstrated the chemotherapeutic and chemopreventive pharmacological effects of the drug. At a dose rate of 20 mg/kg of body weight, TAM induced protection against the infection in mice. In the clinical efficacy studies, a reduction in cyst weight was observed after the administration of 20 mg/kg in mice with cysts developed during 3 or 6 months, compared to that of those collected from control mice. Since the collateral effects of high TAM doses have been largely documented in clinical trials, the use of low doses of this drug as a short-term therapy may be a novel alternative approach for human cystic echinococcosis treatment.Fil: Nicolao, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biologia. Laboratorio de Zoonosis Parasitarias; ArgentinaFil: Elissondo, María Celina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biologia. Laboratorio de Zoonosis Parasitarias; ArgentinaFil: Denegri, Guillermo Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biologia. Laboratorio de Zoonosis Parasitarias; ArgentinaFil: Goya, Alejandra Beatriz. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; ArgentinaFil: Cumino, Andrea Carina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biologia. Laboratorio de Zoonosis Parasitarias; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Química; Argentin

Echinococcus multilocularis: An emerging pathogen in Hungary and Central Eastern Europe?

Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, is reported for the first time in Red Foxes (Vulpes vulpes) in Hungary. This parasite may be spreading eastward because the population of foxes has increased as a consequence of human interventions, and this spread may result in the emergence of alveolar echinococcosis in Central Eastern Europe

Factors influencing the development and carbohydrate metabolism of Echinococcus granulosus in dogs

Echinococcus granulosus adult worms, 35 days postinfection, were measured for dispersion in the intestines of 10 dogs, a range of morphological characters, and the excreted end products of carbohydrate catabolism following 4 hr incubation in vitro. Most worms were found in the proximal sections of the small intestine, but the pattern of dispersion differed between dogs. Worm development varied both between dogs and between different regions of the small intestine of individual dogs. Overall there was a high level of variability with no simple patterns. Worm metabolism was related to worm development and, also independently, to local population density within the intestine. Larger, more mature worms produced less lactate and, at higher densities. worms tended to produce more acetate and succinate (pathways with a higher energy yield than lactate) and less ethanol. Thus, both more developed worms and high population density are associated with a shift from cytosolic to mitochondrial metabolism. The variation between worm populations along the small intestine along with the observed variation between worm populations from sibling dogs infected with genetically identical parasites suggests that the local host environment has a significant effect on parasite development

Post-treatment follow-up study of abdominal cystic echinococcosis in Tibetan communities of northwest Sichuan Province, China

Background: Human cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, with the liver as the most frequently affected organ, is known to be highly endemic in Tibetan communities of northwest Sichuan Province. Antiparasitic treatment with albendazole remains the primary choice for the great majority of patients in this resource-poor remote area, though surgery is the most common approach for CE therapy that has the potential to remove cysts and lead to complete cure. The current prospective study aimed to assess the effectiveness of community based use of cyclic albendazole treatment in Tibetan CE cases, and concurrently monitor the changes of serum specific antibody levels during treatment. Methodology/Principal Findings: Ultrasonography was applied for diagnosis and follow-up of CE cases after cyclic albendazole treatment in Tibetan communities of Sichuan Province during 2006 to 2008, and serum specific IgG antibody levels against Echinococcus granulosus recombinant antigen B in ELISA was concurrently monitored in these cases. A total of 196 CE cases were identified by ultrasound, of which 37 (18.9%) showed evidence of spontaneous healing/involution of hepatic cyst(s) with CE4 or CE5 presentations. Of 49 enrolled CE cases for treatment follow-up, 32.7% (16) were considered to be cured based on B-ultrasound after 6 months to 30 months regular albendazole treatment, 49.0% (24) were improved, 14.3% (7) remained unchanged, and 4.1% (2) became aggravated. In general, patients with CE2 type cysts (daughter cysts present) needed a longer treatment course for cure (26.4 months), compared to cases with CE1 (univesicular cysts) (20.4 months) or CE3 type (detached cyst membrane or partial degeneration of daughter cysts) (9 months). In addition, the curative duration was longer in patients with large (.10 cm) cysts (22.3 months), compared to cases with medium (5– 10 cm) cysts (17.3 months) or patients with small (,5 cm) cysts (6 months). At diagnosis, seven (53.8%) of 13 cases with CE1 type cysts without any previous intervention showed negative specific IgG antibody response to E. granulosus recombinant antigen B (rAgB). However, following 3 months to 18 months albendazole therapy, six of these 7 initially seronegative CE1 cases sero-converted to be specific IgG antibody positive, and concurrently ultrasound scan showed that cysts changed to CE3a from CE1 type in all the six CE cases. Two major profiles of serum specific IgG antibody dynamics during albendazole treatment were apparent in CE cases: (i) presenting as initial elevation followed by subsequent decline, or (ii) a persistent decline. Despite a decline, however, specific antibody levels remained positive in most improved or cured CE cases. Conclusions: This was the first attempt to follow up community-screened cystic echinococcosis patients after albendazole therapy using ultrasonography and serology in an endemic Tibetan region. Cyclic albendazole treatment proved to be effective in the great majority of CE cases in this resource-poor area, but periodic abdominal ultrasound examination was necessary to guide appropriate treatment. Oral albendazole for over 18 months was more likely to result in CE cure. Poor drug compliance resulted in less good outcomes. Serology with recombinant antigen B could provide additional limited information about the effectiveness of albendazole in CE cases. Post-treatment positive specific IgG antibody seroconversion, in initially seronegative, CE1 patients was considered a good indication for positive therapeutic efficacy of albendazole

The Echinococcus canadensis (G7) genome: A key knowledge of parasitic platyhelminth human diseases

Background: The parasite Echinococcus canadensis (G7) (phylum Platyhelminthes, class Cestoda) is one of the causative agents of echinococcosis. Echinococcosis is a worldwide chronic zoonosis affecting humans as well as domestic and wild mammals, which has been reported as a prioritized neglected disease by the World Health Organisation. No genomic data, comparative genomic analyses or efficient therapeutic and diagnostic tools are available for this severe disease. The information presented in this study will help to understand the peculiar biological characters and to design species-specific control tools. Results: We sequenced, assembled and annotated the 115-Mb genome of E. canadensis (G7). Comparative genomic analyses using whole genome data of three Echinococcus species not only confirmed the status of E. canadensis (G7) as a separate species but also demonstrated a high nucleotide sequences divergence in relation to E. granulosus (G1). The E. canadensis (G7) genome contains 11,449 genes with a core set of 881 orthologs shared among five cestode species. Comparative genomics revealed that there are more single nucleotide polymorphisms (SNPs) between E. canadensis (G7) and E. granulosus (G1) than between E. canadensis (G7) and E. multilocularis. This result was unexpected since E. canadensis (G7) and E. granulosus (G1) were considered to belong to the species complex E. granulosus sensu lato. We described SNPs in known drug targets and metabolism genes in the E. canadensis (G7) genome. Regarding gene regulation, we analysed three particular features: CpG island distribution along the three Echinococcus genomes, DNA methylation system and small RNA pathway. The results suggest the occurrence of yet unknown gene regulation mechanisms in Echinococcus. Conclusions: This is the first work that addresses Echinococcus comparative genomics. The resources presented here will promote the study of mechanisms of parasite development as well as new tools for drug discovery. The availability of a high-quality genome assembly is critical for fully exploring the biology of a pathogenic organism. The E. canadensis (G7) genome presented in this study provides a unique opportunity to address the genetic diversity among the genus Echinococcus and its particular developmental features. At present, there is no unequivocal taxonomic classification of Echinococcus species; however, the genome-wide SNPs analysis performed here revealed the phylogenetic distance among these three Echinococcus species. Additional cestode genomes need to be sequenced to be able to resolve their phylogeny.Fil: Maldonado, Lucas Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Assis, Juliana. Fundación Oswaldo Cruz; BrasilFil: Gomes Araújo, Flávio M.. Fundación Oswaldo Cruz; BrasilFil: Salim, Anna C. M.. Fundación Oswaldo Cruz; BrasilFil: Macchiaroli, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Cucher, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Camicia, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Fox, Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Rosenzvit, Mara Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Oliveira, Guilherme. Instituto Tecnológico Vale; Brasil. Fundación Oswaldo Cruz; BrasilFil: Kamenetzky, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentin

A molecular survey of Echinococcus granulosus sensu lato in central-eastern Europe

Central-eastern Europe is an endemic region for cystic echinococcosis where multiple species of intermediate hosts are commonly infected with Echinococcus granulosus sensu lato tapeworms of major medical and veterinary importance. Investigations of the genetic variation of 25 Echinococcus isolates from five countries (Slovakia, Romania, Ukraine, Hungary, Poland) were undertaken using three mitochondrial DNA markers. The 18 isolates from pigs derived from Slovakia and Ukraine and the four human isolates from Slovakia, Poland and Ukraine were identified as E. canadensis G7, whereas the three human isolates from Romania and Hungary were classified as E. granulosus sensu stricto G1. This study reports the first confirmed human case of E. granulosus s.s. in Hungary. The haplotype G7A with two polymorphic sites relative to the most common regional variant of E. canadensis G7 was recorded in both pigs from Ukarine and in a single pig isolate from Slovakia. The results of this study support the circumstantial evidence that E. canadensis G7 with low infectivity for humans is highly prevalent in the northern parts of the region (Poland, Slovakia, forest-steppe zone of Ukraine),while infections with E. granulosus s.s. which are highly infectious for humans are more commonly encountered in Romania and Hungary

Molecular and morphological characterization of Echinococcus granulosus of human and animal origin in Iran

Iran is an important endemic focus of cystic hydatid disease (CHD) where several species of intermediate host are commonly infected with Echinococcus granulosus. Isolates of E. granulosus were collected from humans and other animals from different geographical areas of Iran and characterized using both DNA (PCR-RFLP of ITS1) and morphological criteria (metacestode rostellar hook dimensions). The sheep and camel strains/genotypes were shown to occur in Iran. The sheep strain was shown to be the most common genotype of E. granulosus affecting sheep, cattle, goats and occasionally camels. The majority of camels were infected with the camel genotype as were 3 of 33 human cases. This is the first time that cases of CHD in humans have been identified in an area where a transmission cycle for the camel genotype exists. In addition, the camel genotype was found to cause infection in both sheep and cattle. Results also demonstrated that both sheep and camel strains can be readily differentiated on the basis of hook morphology alone

Echinococcus, Giardia and Cryptosporidium: observational studies challenging accepted dogma

The development of in vitro culture systems that allow the maintenance, and support the development of Echinococcus, Giardia and Cryptosporidium in the laboratory have had a significant impact on their biology and taxonomy and the epidemiology of infections they cause. This short retrospective review demonstrates how radical shifts in our understanding have occurred as a result of being able to grow these organisms in culture, and how molecular tools have helped in the interpretation of such research that often reflects the observations of earlier workers