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.

Babesia canis vogeli infection in dogs and ticks in the semiarid region of Pernambuco, Brazil

Abstract:This study aimed to report the prevalence of Babesia canis vogeli in dogs and ticks in the urban and rural areas of Petrolina, Pernambuco. Serum and peripheral blood samples of 404 dogs were tested by indirect immunofluorescence assay (IFA) and by blood smears, respectively. The presence of tick infestation was evaluated, and some specimens were submitted to DNA amplification by polymerase chain reaction (PCR). The presence of antibodies anti-B. canis vogeli was determinate in 57.9% (234/404) of dogs. The direct detection of Babesia spp was obtained in 0.5% (2/404) dogs by visualization of intraerythrocytic forms. Infestation by Rhipicephalus sanguineus sensu lato was observed in 54.5% (220/404) of dogs in both urban and rural areas. DNA of Babesia canis vogeli were obtained by PCR in 6% individual (3/50) and 8.7% of pool of ticks (7/80). The risk factors for the presence of anti-B. canis vogeli antibodies, as determined through the application of logistic regression models (P<0.05), were the following: medium breed size variables (P<0.001); contact with areas of forest (P=0.021); and access on the street (P=0.046). This study describes, for the first time, the confirmation of infection of B. canis vogeli in dogs and ticks in the semiarid region of Pernambuco, Brazil.

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.

Molecular and serological detection of Ehrlichia canis and Babesia vogeli in dogs in Colombia

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

A Review of the Genus Echinococcus Rudolphi, 1801

1. The genus Echinococcus Rudolphi, 1801, is reviewed. The recorded measurements of all species are shown in tables, and their taxonomic status is discussed. 2. Three species of Echinococcus can be distinguished by morphological criteria and are considered valid: E. granulosus, E. multilocularis and E. oligarthrus. The status of E. felidis and E. patagonicus is uncertain, but both may be conspecific with E. granulosus. Six species are considered synonyms of E. granulosus: E. cameroni, E. intermedius, E. longimanubrius, E. lycaontis, E. minimus and E. ortleppi.

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.

On the Ecology and Distribution of Echinococcus spp. (Cestoda: Taeniidae), and Characteristics of Their Development in the Intermediate Host. II. Comparative Studies on the Development of Larval E. multilocularis Leuckart, 1863, in the Intermediate Host

This paper reports the results of a comparative study of the development of the larval Echinococcus multilocularis Leuckart, 1863), and associated tissue reaction in naturally and experimentally infected mammals representing 31 species. The histogenesis of the larval cestode was traced in detail in arvicoline rodents of several species, and interspecific differences were defined. In arvicoline rodents, the developing larva exhibited host-specific characteristics within about a month after infection was established. The tissue reaction in Microtus oeconomus was characterized by the production of a large quantity of detritus around the larva, and by the formation of a thick epithelioid zone. In one subspecies, M. oeconomus innuitus, development of the larva was retarded, and the detrital mass was often calcified; in another, M. oeconomus operarius, the detritus rarely became calcified and the larva proliferated more rapidly. In M. pennsylvanicus, the tissue reaction was minimal, and little detritus was present. The characteristics of the tissue reaction in M. montebelli placed it in an intermediate position between the aforementioned species. In Clethrionomys rutilus, a thin epithelioid zone and an outer zone of loose collagenous fibers composed the adventitial layer; exogenous budding was retarded in this vole. A minimal tissue reaction occurred in Lagurus curtatus. In Lemmus spp., larger cysts were characteristic, but areas of small-cystic proliferation were always present. Similar differences in species or subspecies of Citellus and Dicrostonyx were described. Lesions of alveolar bydatid disease in man also were studied. The invasive growth of the larval cestode in the human liver involves a process comparable to small-cystic proliferation in the natural intermediate hosts. Although the later stages of development of the larval cestode are inhibited in man, exogenous proliferation of vesicles continues for the life of the host. The lesion in man was compared with a morphologically similar formation produced by anomalous development of the larval E. granulosus in the bovine liver. The latter is distinguished by the absence of areas of small-cystic proliferation. Non-echinococcal lesions found in the tissues studied, some of which resembled foci caused by the larval E. multilocularis, were briefly discussed.

Taxonomic Characters in the Genus Echinococcus (Cestoda: Taenlidae)

Not more than four species of cestodes of the genus Echinococcus Rudolphi, 1801, are recognized as valid. The larval stage of at least three of them is able to develop in man, causing respective types of hydatid disease. Accurate characterization of these cestodes, including both larval and adult stages, is essential for identification, upon which depends development of methods for preventing infection of man and domestic animals. Because morphological characteristics of the larval cestodes may be modified according to the species of host in which they develop, identification should be based upon taxonomic characters of specimens from the respective natural hosts, which can be identified by means of ecological investigations in endemic areas in combination with controlled infection of experimental animals. The morphological and biological characteristics of the known species are discussed, and the two species most important to public health and economically--E. granulosus (Batsch, 1786) and E. multilocularis Leuckart, 1863--are distinguished. French résumé: Trois espèces de cestodes du genre Echinococcus Rudolphi, 1801 ont été identifiées: E. granulosus (Batsch, 1786); E. multilocularis Leuckart, 1863; et E. oligarthrus (Diesing, 1863). On n'a pas encore déterminé avec certitude s'il fallait leur adjoindre E. patagonicus Szidat, 1960. Ce dernier mis à part, tous ces ténias sont connus pour provoquer chez l'homme des formes spécifiques d'échinococcose hydatique; l'infestation par les larves de E. granulosus est également fréquente chez les ongulés domestiques et la répartition de ce cestode est presque cosmopolite. E. multilocularis est très largement répandu dans l'hémisphère nord, tandis que E. oligarthrus ne se trouve que dans le centre et le sud de I'Amérique. Pour définir des critères morphologiques qui permettent de distinguer avec précision les différentes espèces d'Echinococcus, il convient de prendre les spécimens sur leurs hôtes naturels, sinon l'évolution du stade strobilaire se trouve habituellement retardée et le stade larvaire présente une gamme étendue de variations morphologiques. Pour identifier les hôtes naturels, il faut étudier l'écologie dans les zones d'éndemicité, ainsi que les manifestations de l'infection contrôlée chez des animaux d'expérience. Le stade strobilaire des ténias peut présenter plusieurs caractères spécifiques importants qui concernent notamment la taille, le nombre et la répartition des testicules, la position du pore génital dans les segments gravides, la forme de l'utérus gravide et, dans certains cas, la taille et la forme des crochets du rostre. La structure de la larve normalement développée est caractéristique de chacune des trois espèces relativement bien connues. Les caractéristiques biologiques jouent également un rôle important dans la différenciation de ces cestodes. Au stade larvaire, le laps de temps nécessaire au développement des scolex infectants est directement fonction de la durée de vie de l'hôte intermédiaire. La larve de E. multilocularis présente des scolex infectants dès le deuxième ou le troisième mois, alors qu'ils n'apparaissent qu'au bout d'un ou deux ans chez la larve de E. granulosus. Un tableau donne la liste comparative des caractéristiques taxonomiques qui permettent de faire la distinction entre ces deux espèces.

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.