Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines.

Therapeutic effects of Sophora moorcroftiana alkaloids in combination with albendazole in mice experimentally infected with protoscolices of Echinococcus granulosus

The objective of the present study was to determine if the combination of alkaloids from Sophora moorcroftiana seeds and albendazole might be effective in the treatment of experimental echinococcosisin female NIH mice (6 weeks old and weighing 18-20 g, N = 8 in each group) infected withprotoscolices of Echinococcus granulosus. Viable protoscolices (N = 6 x 103) were cultured in vitro in 1640 medium and mortality was calculated daily. To determine the in vivo efficacy, mice were inoculated intraperitoneally with viable protoscolices and then treated once daily by gavage for three months with the alkaloids (50 mg kg-1 day-1) and albendazole (50 mg kg-1 day-1), separately and in combination (both alkaloids at 25 mg kg-1 day-1 and albendazole at 25 mg kg-1 day-1). Next, the hydatid cysts collected from the peritoneal cavity of the animals were weighed and serum IL-4, IL-2, and IgE levels were analyzed. Administration of alkaloids to cultured protoscolices showed significant dose- and time-dependent killing effects. The weight of hydatid cysts was significantly decreased upon treatment with each drug (P < 0.01), but the decrease was more prominent and the rate of hydatid cyst growth inhibition was much higher (76.1%) in the group receiving the combined treatments (18.3 ± 4.6 mg). IL-4 and total IgE were decreased (939 ± 447 pg/mL and 2.03 ± 0.42 IU/mL, respectively) in serum from mice treated with alkaloids and albendazole compared with the untreated control (1481 ± 619 pg/mL and 3.31 ± 0.37 IU/mL; P < 0.01). These results indicate that S. moorcroftiana alkaloids have protoscolicidal effects and the combination of alkaloids and albendazole has significant additive effects.

Echinococcus granulosus : determinación de la respuesta inmunecelular de los antígenos principales libres y atrapados en liposomas en hidatidosis experimental

Tesis (Maestría en Ciencias con Especialidad en Inmunobiología) UANL

Echinococcus granulosus : participación de citocinas en la regulación de la respuesta inmune en Hidatidosis experimental

Tesis (Doctorado en Ciencias con Especialidad en Microbiología) UANL

Isolamento e caracterização de microssatélites do genoma de Echinococcus granulosus (Cestoda, Taeniidae)

A presença de microssatélites no genoma de Echinococcus granulosus foi verificada utilizando-se oito oligonucleotídeos com repetições como sondas (GT15, CT15, AT15, CG15, CAT10, CAA10, CGG10 e CATA10). Experimentos de hibridização revelaram que as repetições GT, CAA, CATA e CT são as mais frequentes no genoma de E. granulosus. As sondas AT e GC não apresentaram sinais de hibridização. Seis lócus contendo repetições CA/GT, quatro lócus contendo repetições GA/CT e oito lócus contendo repetições AAC/GGT foram clonados e sequenciados. O lócus Egmsca1 foi analisado em 73 isolados do Brasil e da Argentina cujas linhagens haviam sido previamente caracterizadas e em 27 isolados provenientes da Etiópia cujas linhagens ainda não foram identificadas. Os isolados brasileiros da linhagem bovina e os isolados argentinos da linhagem do camelo apresentaram-se monomórficos e compartilharam o alelo (CA)7. Isolados argentinos das linhagens da ovelha e da ovelha da Tasmânia compartilharam dois alelos [(CA)8 e (CA)10] com os isolados brasileiros da linhagem da ovelha. O alelo (CA)11 foi encontrado somente em isolados brasileiros da linhagem da ovelha em uma baixa frequência. O alelo (CA)9 ocorreu apenas em um isolado da Etiópia. As populações brasileira e argentina da linhagem da ovelha foram testadas em relação ao equilíbrio de Hardy-Weinberg e somente a primeira estava de acordo com as expectativas. Protoescólices isolados de um único cisto hidático não apresentaram polimorfismo, validando a utilização de protoescólices de um mesmo cisto, agrupados como um isolado, em estudos populacionais. Um microssatélite contendo repetições de pentanucleotídeos, contido no complexo gênico do snRNA U1, também foi isolado. Este estudo descreve pela primeira vez o isolamento e caracterização de microssatélites em E. granulosus.

Hidatidose policística no Estado do Acre: contribuição para o diagnóstico, tratamento e prognóstico dos pacientes

A equinococose é uma zoonose cujos agentes etiológicos são helmintos do gênero Echinococcus. Há cinco espécies de Echinococcus, duas delas, o E. oligarthrus (Diesing, 1863) e o E. vogeli (Rausch & Bernstein, 1972) ocorrem apenas em zonas neotropicais. A equinococose pelo E. vogeli provoca cistos hidáticos múltiplos, principalmente no fígado dos hospedeiros intermediários, dos quais um deles é o ser humano. O pouco conhecimento acerca da doença faz com que o diagnóstico seja retardado ou até mesmo equivocado. A falta de sistematização nas indicações de tratamento também dificulta a avaliação dos resultados e prognóstico dos pacientes com lesões hepáticas e peritoneais causadas pelo E. vogeli. Neste trabalho, descrevemos o quadro clínico dos pacientes; propomos protocolo de classificação radiológica, utilizado na classificação da equinococose alveolar (E. multilocularis, Classificação “PNM”, Kern et al., 2006), que foi adequado também para a equinococose policística (E. vogeli); e descrevemos uma opção terapêutica para o tratamento dessa hidatidose que anteriormente só havia sido utilizada para casos de equinococose cística (E. granulosus, PAIR -Puncture, Aspiration, Injection, Reaspiration, Brunnetti et al., 2001). Uma coorte prospectiva foi iniciada no ano de 1999 e até 2009 foram incluídos 60 pacientes. Foram descritos os principais sintomas e sinais: dor no andar superior do abdome (65%) e hepatomegalia (60%) e os pacientes foram classificados conforme a Classificação “PNM” e submetidos a três modalidades terapêuticas: (i) quimioterapia com albendazol na dose de 10mg/Kg/dia, (ii) tratamento cirúrgico com ressecção dos cistos ou (iii) punção percutânea – PAIR. Após exclusão de 2 casos, por preenchimento inadequado do protocolo de pesquisa, os grupos foram assim distribuídos: terapêutica com albendazol: n=28 (48,3%; 28/58), terapêutica cirúrgica: n=25 (52,1%; 25/58) e PAIR: n=5 (8,1%; 5/58). Os resultados foram estratificados conforme o resultado da terapêutica: “Cura”, representada pelo desaparecimento das lesões após tratamento clínico ou cirúrgico; “Melhora clínica”, entendidas como pacientes assintomáticos, sem perda ponderal e com as funções fisiológicas preservadas; “Sem Melhora”, incluiu os pacientes que permaneceram sintomáticos; “Óbito”; e “Sem informação”, o acompanhamento não permitiu a conclusão sobre o desfecho. Nos três grupos terapêuticos a taxa de letalidade de 15,5% (9/58), “sem melhora” 1,7% (1/58), “melhora clínica” em 40,0% (23/58) e “cura” em 32,8% (19/58). Com relação ao desfecho “óbito”, não houve diferença entre as terapêuticas com albendazol ou cirúrgica com 4 (14,2%) e 3 (12%) óbitos respectivamente; porém, no primeiro grupo, albendazol, o desfecho “cura” foi de 4,3% (1/23) e “melhora clínica” 74,0% (17/23), enquanto que no grupo “cirurgia” a “cura” representou 71,0% (17/24) e “melhora clínica” com 16,7(4/24). A terapêutica “PAIR” foi associado a taxa de letalidade de 40% (2/5), cura em 20% (1/5) e melhora clínica em 40% (2/5). A Classificação “PNM” foi útil para indicar tipo terapêutica nos casos de hidatidose policística. Em conclusão, na série estudada a terapêutica cirúrgica apresenta melhor resultado que a terapêutica clínica quanto aos desfechos “cura” e “melhora clínica”. A terapêutica por PAIR necessita de mais estudos.

Echinococcus Infections in Man and Animals in Kenya

1. Hydatid cysts are found in more than 30 per cent of all cattle, sheep and goats in Kenya, but the disease is prevalent in man only in the semi-desert area of Turkana. Up to the time of the present investigation the life-cycle of the parasite in East Africa had not been studied, but it was suggested that wild carnivores, such as hyenas and jackals, might be the main hosts of the adult worms. 2. One hundred and forty-three carnivores, representing 23 species, have been examined. Echinococcus adults were found in 27 out of 43 domestic dogs (Canis familiaris), in three out of four hunting dogs (Lycaon pictus), in one out of nine jackals (Thos mesomelas), and in three out of 19 hyaenas (Crocuta crocuta). 3. A detailed morphological study was made of the Kenya material. After comparison with specimens from other parts of the world, it was concluded that the only species occurring in Kenya was E. granulosus, but it is possible that the minor morphological and biological differences are evidence of distinct strains. Further laboratory studies are necessary to compare the parasite from man and animals in different parts of Kenya with material from elsewhere. 4. A search was made for larval hydatids in 92 ungulates representing 18 species, and in a miscellaneous collection of nearly 2,000 rodents and primates representing a further 31 species. Only one animal was positive, a wildebeest (Gorgon taurinus). 5. The infections in the wild carnivores were all very light; only domestic dogs were heavily infected. It is concluded that the main cycle of transmission in Kenya is between dogs and domestic livestock. 6. Turkana tribesmen are the most heavily infected people in Kenya, either because the strain of parasite is more pathogenic to man in that area, or, more probably, because of the intimate contact between children and the large number of infected dogs. A particularly dangerous custom in the area is the use of dogs to clean the face and anal regions of babies when they vomit or have diarrhea. No satisfactory explanation can be given for the rarity of the disease in man in many of the other areas of Kenya where hydatids are very common in domestic animals. 7. The control of the disease will depend upon an active health-education campaign, together with the destruction of all unregistered dogs and improvement in meat hygiene.

Studies on the Helminth Fauna of Alaska. XXV. The Ecology and Public Health Significance of Echinococcus sibiricensis Rausch & Schiller, 1954, on St. Lawrence Island

The occurrence of a species of Echinococcus (Rudolphi, 1801) on St Lawrence Island was noted by the writers in early 1950. Recognition of its unusual host relationships led to an investigation of the ecology of this cestode, E. sibiricensis Rausch & Schiller, 1954. It is the purpose of this paper to report the results of this work, with emphasis on alveolar hydatid disease in man, of which this cestode is the etiologic agent.

The Taxonomic Value and Variability of Certain Structures in the Cestode Genus Echinococcus (Rudolphi, 1801) and a Review of Recognized Species

Although cestodes of the genus Echinococcus have been much studied in the past, there is need for an evaluation of these morphological characters used as the basis for species differentiation. The generous cooperation of other investigators in providing necessary foreign material and the results of nearly five years of field work in Alaska make possible such a study. It is the purpose of the paper to evaluate morphological characters used at the species level to differentiate these cestodes, and to review the status of species currently considered valid.

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.

Taxonomic position and geographical distribution of the common sheep G1 and camel G6 strains of Echinococcus granulosus in three African countries

The taxonomic and phylogenetic status of Echinococcus granulosus strains are still controversial and under discussion. In the present study, we investigated the genetic polymorphism of E. granulosus isolates originating from three countries of Africa, including a region of Algeria, where the common G1 sheep and the camel G6 strains coexist sympatrically. Seventy-one hydatid cysts were collected from sheep, cattle, camels, and humans. Two mitochondrial markers (cox1 and nad1) were used for strain identification. Two nuclear markers (actII and hbx2) were used to study the possible occurrence of cross-fertilization. Despite the heterogeneity observed among the G1 isolates, they were all localized within one robust cluster. A second strong cluster was also observed containing all of the G6 isolates. Both strains appeared as two distinct groups, and no cases of interbreeding were found. Thus, the attribution of a species rank can be suggested. We also found the Tasmanian sheep G2 strain for the first time in Africa. Because of the slight variations observed between the common sheep and the Tasmanian sheep strains, further studies should be carried out to elucidate the epidemiological relevance of this genetic discrimination.

Isolation and molecular characterization of recombinant Echinococcus granulosus P29 protein (recP29) and its assessment for the post-surgical serological follow-up of human cystic echinococcosis in young patients

We synthesized recombinant Echinococcus granulosus protoscolex recP29 antigen to be preliminarily assessed by ELISA and immunoblotting. RecP29-serology was carried out on 54 young patients with cystic echinococcosis (CE). Patients were classified into either cured (CCE) (n=40) or non-cured (NCCE) (n=14) CE patients. RecP29 ELISA showed a gradual decrease of antibody concentrations in all CCE cases that were initially (before treatment) seropositive to this antigen (25 out of 40) or that seroconverted following treatment. A complete seronegativity was reached within 3 years post-surgery in all of these cases. Conventional HCF ELISA yielded seronegativity in only 10% of initially recP29-seropositive CCE patients (P=0.086). Likewise, recP29 immunoblotting yielded seronegativity in 93% of 29 out of 40 initially recP29-immunoblot-positive CCE patients after 3 years follow-up, compared with 72% in the HCF immunoblotting (P=0.060). Eleven out of 14 NCCE patients were initially positive by recP29 ELISA, and 10 out of these maintained a marked anti-recP29 antibody reactivity until the endpoint of the follow-up period. All 14 NCCE cases were initially seropositive by recP29 immunoblotting, and 13 cases remained seropositive until the end of the study. Thus, recombinant P29 protein appears prognostically useful for monitoring those post-surgical CE cases with an initial seropositivity to this marker.

The EG95 antigen of Echinococcus spp. contains positively selected amino acids, which may influence host specificity and vaccine efficacy

Echinococcosis is a worldwide zoonotic parasitic disease of humans and various herbivorous domestic animals (intermediate hosts) transmitted by the contact with wild and domestic carnivores (definitive hosts), mainly foxes and dogs. Recently, a vaccine was developed showing high levels of protection against one parasite haplotype (G1) of Echinococcus granulosus, and its potential efficacy against distinct parasite variants or species is still unclear. Interestingly, the EG95 vaccine antigen is a secreted glycosylphosphatydilinositol (GPI)-anchored protein containing a fibronectin type III domain, which is ubiquitous in modular proteins involved in cell adhesion. EG95 is highly expressed in oncospheres, the parasite life cycle stage which actively invades the intermediate hosts. After amplifying and sequencing the complete CDS of 57 Echinococcus isolates belonging to 7 distinct species, we uncovered a large amount of genetic variability, which may influence protein folding. Two positively selected sites are outside the vaccine epitopes, but are predicted to alter protein conformation. Moreover, phylogenetic analyses indicate that EG95 isoform evolution is convergent with regard to the number of beta-sheets and alpha-helices. We conclude that having a variety of EG95 isoforms is adaptive for Echinococcus parasites, in terms of their ability to invade different hosts, and we propose that a mixture of isoforms could possibly maximize vaccine efficacy.