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Immunoelectrophoresis tests showing Echinococcus granulosus arc 5 in human cases of Echinococcus vogeli and cysticercosis-multiple myeloma.

Human sera from one case of polycystic hydatidosis due to Echnincoccus vogeli and from a case of cysticercosis and multiple myeloma were positive to the immunoelectrophoresis (IEP) test for hydatidosis based on the E. granulosus arc 5 positivity criterion. Arc 5 can therefore be elicited in IEP tests of human sera not only by E. granulosus and E. multilocularis but also by E. vogeli antigens. Whether the cross reaction observed in the second serum was due to the multiple myeloma or to the cysticerci remains to be determined. Although arc 5 antigens are known to be present in Taenia hydatigena cyst fluid this is the first report of an arc 5 due to antigens other than Echinococcus in IEP of human sera.

Adult

Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing.

The pattern of species and strain variation within the genus Echinococcus is complex and controversial. In an attempt to characterise objectively the various species and strains, the sequence of a region of the mitochondrial cytochrome c oxidase subunit I (CO1) gene was determined for 56 Echinococcus isolates. Eleven different genotypes were detected, including 7 within Echinococcus granulosus, and these were used to categorise the isolates. The 4 generally accepted Echinococcus species were clearly distinguishable using this approach. In addition, the consensus view of the strain pattern within E. granulosus, based on a variety of criteria of differentiation, was broadly upheld. Very little variation was detected within Echinococcus multilocularis. Remarkable intra-strain homogeneity was found at the DNA sequence level. This region of the rapidly evolving mitochondrial genome is useful as a marker of species and strain identity and as a preliminary indication of evolutionary divergence within the genus Echinococcus.

Amino Acid Sequence

Sequencing and characterization of an Echinococcus multilocularis DNA probe and its use in the polymerase chain reaction.

The nucleotide sequence of the cloned Echinococcus multilocularis DNA probe pAL1 was determined in order to simplify and improve the sensitivity of a diagnostic assay through the application of the polymerase chain reaction (PCR). The insert-specific oligonucleotides BG1 and BG2 define a 2.6-kb fragment in the genomic DNA of E. multilocularis, while BG1 and BG3 define a 0.3 kb fragment. A PCR study including 14 independent E. multilocularis isolates in addition to Echinococcus granulosus. Echinococcus vogeli, Taenia spp. and other cestodes revealed that the 2.6-kb fragment was amplified from genomic DNA of all E. multilocularis isolates tested (originating from Switzerland, Alaska, Canada, France, Germany and Japan), but from genomic DNA of none of the other cestode species. PCR with BG1 and BG2 furthermore uniquely resulted in the synthesis of a 0.55-kb fragment specific for Taenia saginata and a 0.6-kb fragment specific for T. taeniaeformis. In contrast to the species specificity of the 2.6-kb BG1/BG2 product, the 0.3 kb (BG1/BG3) product demonstrated genus specificity: the 0.3-kb product was amplified from genomic DNA of all E. multilocularis, E. granulosus and E. vogeli isolates tested, but from genomic DNA of none of the other cestode species. The diagnostic sensitivity of PCR using both primer sets was determined to be 50 pg parasite DNA, suggesting the practical utility of this simple assay in demonstrating parasite DNA in specimens from a variety of sources. At the basic level, the pAL1-derived oligonucleotides may also prove useful in assessing strain variation, RFLPs or other manifestations of genetic variation in E. multilocularis.

Animals

Release and survival of Echinococcus eggs in different environments in Turkana, and their possible impact on the incidence of hydatidosis in man and livestock.

In Turkana, Kenya, a prevalence of hydatidosis of nearly 10% has been recorded among the pastoralists yet their livestock have a much lower prevalence of the disease. The present study investigated the release from dogs and subsequent survival of Echinococcus eggs in Turkana huts, water-holes and in the semi-arid environment. The results were compared with the survival of eggs of Taenia hydatigena and T. saginata. The study was repeated under the cooler and moister conditions found in Maasailand where livestock have a greater incidence of hydatid disease than in Turkana but where the incidence in man is ten times lower. The average number of Echinococcus eggs per proglottid is 823. Nine percent of these remain in proglottids 15 minutes after release from a dog and the released eggs lose their viability in less than two, 48 and 300 hours in the sun, huts and water in Turkana respectively: the major influencing factor being temperature. The greater survival of eggs in the houses, coupled with the fact that dogs congregate for most of the day in the small houses facilitating a close man:dog contact, provide ideal conditions for the transmission of the parasite to man. The hostile environmental conditions and lack of contact between dogs and livestock contributes to the lower infection rate in livestock. Conversely in Maasailand, Echinococcus eggs survive in the environment for longer than three weeks and in addition, dogs are used for herding. This partly explains the higher infection rate among Maasai livestock but the low human infection rate remains arcane and requires further study.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance

The role of foxes Vulpes vulpes in the epidemiology of Echinococcus granulosus in urban environments.

OBJECTIVE: To survey the prevalence of intestinal worms, particularly Echinococcus granulosus, in foxes in Canberra. DESIGN: The locations of foxes seen in Canberra during this study were recorded. Foxes and macropod marsupials killed on the roads of Canberra were collected and examined for the presence of intestinal helminths and hydatid cysts respectively. METHOD: The intestinal contents of the foxes were washed through a fine sieve and examined microscopically. All helminths recovered were collected and identified. All the internal organs of the macropods were examined for any cystic lesions. RESULTS: Forty-five foxes and 44 macropods were examined. Echinococcus granulosus was found in three of the foxes (7%). Hydatid cysts were not found in the internal organs of any of the macropods examined. CONCLUSIONS: Echinococcus granulosus is present in the urban fox population of Canberra. This hitherto unreported aspect of the epidemiology of E. granulosus in Australia could be a potential public health risk to urban populations.

Animals

[Cryosurgery in the treatment of Echinococcus alveolaris. Animal experiments].

The influence of cryosurgery on echinococcus alveolaris is studied in 100 mice. In a second series cryosurgical effects on structures of the hepatic portal (hepatic artery, portal vein, hepatic duct) were examined. The experiments showed that echinococcus alveolaris can be devitalized by cryosurgical measures at temperatures between -100 degrees C and -190 degrees C. No damage of the hepatic artery, portal vein, and hepatic duct occurred. Clinical application of cryosurgery for the treatment of inoperable echinococcus alveolaris of the hepatic portal appears to be justified.

Animals

Detection of Echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Echinococcus coproantigens in fecal samples from dogs, dingoes or foxes infected with either E. granulosus or E. multilocularis. The ELISA was based on protein-A-purified polyclonal antibodies [anti-E. granulosus excretory/secretory (E/S) antigens]. The specificity of the assay as determined in 155 samples derived from carnivores that were free of helminth infection (n = 37) or infected with non-Echinococcus cestodes (n = 76) or with various nematodes (n = 42) was found to be 98% overall. The diagnostic sensitivity was strongly dependent on the homologous worm burden. All 13 samples from foxes harboring greater than 1,000 E. multilocularis worms and 13 of 15 (87%) samples from dogs or dingoes containing greater than 200 E. granulosus worms were ELISA-positive, whereas 34 of 46 samples from foxes harboring less than 1,000 E. multilocularis and 9 of 10 samples from dogs or dingoes bearing less than 200 E. granulosus tested negative. Experimental prepatent infections of dogs with E. granulosus revealed positive ELISA reactions within the prepatent period (10-20 days post-infection) for six animals bearing greater than 1,000 E. granulosus each; a low worm burden (less than 1,000 tapeworms/animal) resulted in ELISA positivity in only 2 of 3 animals at 30 days post-infection at the earliest. All five dogs that had been experimentally infected with E. multilocularis tested positive in the coproantigen ELISA as early as on day 5 post-infection.

Animals

[Diagnosis and therapy of liver echinococcus (author's transl)].

From 1970 to April 1975 42 patients were treated for Echinococcus of the liver at the Bonn University Dept. of Surgery. There were 33 cases of E. cysticus and 9 cases of E. alveolaris. These two types of Echinococcus, different in parasitology and epidemiology present different clinical manifestation of disease with different course and prognosis. Angiography with celiaco- and superselective hepaticography are decisive for diagnosis. Therapy can only be surgical with total removal of the parasites. In E. cysticus this is almost always possible by enucleation-resection or pericystectomie following evacuation of the cyst and instillation of 20% sodium-chloride or formaldehyde. Such radicality is the exception in E. alveolaris. Here partial resections, biliodigestive and hepatodigestive anastomoses as palliative measures are carried out predominantly to ensure bile passage.

Adult

Cloning and characterisation of an immunodominant major surface antigen of Echinococcus multilocularis.

A lambda gt11 cDNA expression library from mRNA of Echinococcus multilocularis protoscolices has been constructed in Escherichia coli Y1090. Immunoscreening with pooled sera obtained from patients suffering from E. multilocularis disease revealed 5 reactive clones. By partial DNA sequence comparison all clones proved to encode the same gene. The complete cDNA sequence of the clone pEM10 with the largest insert of 2.2 kb was determined and an open reading frame of 1.7 kb could be described. The derived amino acid sequence shares 42.6% identity with human microvillar cytovillin found in the membranes of placenta and carcinoma tissues. The coding region of the cDNA of pEM10 was amplified by polymerase chain reaction (PCR) and cloned in frame into expression vector pGEX-3X. Immunoblot analysis revealed the expression of a recombinant antigen of 65 kDa and a protein with the same molecular weight was also found in the lysate of E. multilocularis protoscolices. In contrast, the protein was absent from hydatid fluid or larvae of Echinococcus granulosus. By means of immunofluorescence studies this immunodominant antigen could be located in the germinal layer of brood capsules and in the tegument of E. multilocularis protoscolices. The fusion protein was purified and used for diagnostic purposes in immunoblot. The diagnostic value of this antigen is discussed.

Amino Acid Sequence

A monoclonal antibody against Echinococcus multilocularis Em2 antigen.

A monoclonal antibody (MAb G11) species-specific to the Em2 antigen of Echinococcus multilocularis was generated for (i) further biological characterization of the Em2 antigen, (ii) easy affinity-purification of Em2 antigen for immunodiagnostic and immunological investigations and (iii) development of a sandwich-ELISA for the detection of Em2 antigen in diagnostic samples and thus species-specific identification of E. multilocularis metacestode material. The MAb G11 was used in an antibody sandwich-ELISA to detect soluble Em2 antigen with a methodical sensitivity of 80 ng E. multilocularis antigen/ml of solution. MAb G11 specifically detected Em2 antigen in all of 15 E. multilocularis-isolates originating from various geographical areas and in none of other helminth isolates (e.g. Echinococcus granulosus, E. vogeli, and others). Further biological analysis by FITC-labelled MAb G11 demonstrated unique binding activity to the laminated layer of the metacestode. Also, oncospheres were binding FITC-labelled MAb G11 on an outer layer synthesized during cultivation in vitro for 13 days after hatching. Application of the MAb G11 antibody sandwich-ELISA for investigation of solubilized oncospheres confirmed the in vitro synthesis of Em2 antigen by oncospheres on day 13 p.i. Adult stages (somatic antigens) and freshly hatched oncospheres were always MAb G11 negative. Solid-phase MAb G11 was used for purification of the corresponding Em2 antigen by affinity chromatography. A preliminary serological evaluation of the Em2(G11) antigen by ELISA revealed identical immunodiagnostic characteristics, compared to Em2 obtained by classical means, thus suggesting the presented method for future isolation of large-scale Em2 antigen.

Animals

The epidemiology of Echinococcus granulosus in the U.K. VIII. The structure of adult colonies of E.g. equinus say in farm dogs in Wales.

The status of Echinococcus granulosus equinus in farm dogs was examined in two valleys in South Powys, Wales. In one valley 76% of the dogs examined were infected, and there was at least one infected dog on every farm except one. In the second valley 49% of the dogs were infected. The structure of natural adult colonies of Echinococcus is described, and the population dynamics of the parasite in the U.K. is discussed. Every colony in the dogs, excepting the very smallest and the very youngest, consists of parasites at several stages of development. Most colonies comprise less than 20 worms. Colony density is greatest about the second or third eighth of the total length of the small intestine. The general characteristics of a colony (other than number) are reflected in the characteristics of the group of worms 'resident' in each successive eighth of the small intestine. Neither the age nor the sex of the host has any obvious effect on the incidence or the size of the colony, its development, or the ultimate infectivity of the colony. The parasites continue to grow throughout life, and growth may be largely independent of the worms' development. As a result, their length is very variable. The number of eggs in each gravid proglottid varies considerably, from 12-1626. All the evidence available and derived from the study of natural populations confirms that E. granulosus is an obligate self-fertilizing hermaphrodite, and that serial development of gravid proglottids does not occur.

Animals

Echinococcus granulosus: specific quantification of the two most immunoreactive antigens in hydatid fluids.

Preparations of the two most immunoreactive Echinococcus granulosus antigens (antigens 4 and 5) from sheep hydatid fluid, purified by a simplified method, and monospecific antisera against antigens 4 and 5, prepared by a new procedure, were used to measure the antigenic concentrations of antigens 4 and 5 in swine, sheep, and human hydatid fluids from pulmonary or hepatic cysts. Two bovine samples and two commercial preparations were also tested. The concentration of both antigens was significantly higher in sheep and human hydatid fluids than in swine hydatid fluid. The antigenic content of the two bovine samples and of the two commercial preparations was below the sensitivity level of the method employed. Independently of the species tested, the amount of Echinococcus antigens was greater in hepatic than in pulmonary cysts. The ratio between the concentrations of antigens 4 and 5 was constant at about 1:10 in the samples from various organs and from different species. When there were enough samples for statistical analysis a linear correlation was found between the contents of these two antigenic components but there was none between the amounts of proteins and the antigenic concentrations in the single cysts. Sheep hydatid fluid must therefore be considered the best source of antigenic material for diagnostic purposes even though in human cysts the antigenic fraction is less contaminated by serum proteins. We describe a reliable method of standardising antigenic material for the immunodiagnosis of hydatid disease.

Animals

Sylvatic echinococcosis in Argentina. I. On the morphology and biology of strobilar Echinococcus granulosus (Batsch, 1786) from domestic and sylvatic animal hosts.

Dogs were fed larvae of Echinococcus granulosus (Batsch, 1786) recovered from sheep in Argentina. The morphological characteristics of strobilae recovered from dogs at 28, 60 and 76 days postexposure were compared with those of Echinococcus spp. recovered from naturally infected foxes, Dusicyon clupaeus (Molina) and D. griseus (Gray), and those of worms reared in dogs from larvae in European hares, Lepus europaeus (L.). Only slight differences were observed in characters of taxonomic importance and it was concluded that all cestodes represented a single species, E. granulosus. The results are discussed as they bear on the speciation controversy and the potential epidemiology importance of Dusicyon spp. as hosts of E. granulosus. It was suggested that E. patagonicus Szidat, 1960 previously described from D. culpaeus is conspecific with E. granulosus. E. cepanzoi Szidat, 1971 is probably also a synonym of E. granulosus and the subspecies E. g. dusicyontis Blood and Lelijveld, 1969, is rejected on the grounds that there is no evidence for host specificity and ecological or other segregation from the nominate form. Foxes appear to become infected from scavenging on dead sheep in localities where E. granulosus is endemic in domestic animals. There is still no evidence that E. granulosus is maintained in Argentina in sylvatic cycles.

Animals

Sylvatic echinococcosis in Argentina. II. Susceptibility of wild carnivores to Echinococcus granulosus (Batsch, 1786) and host-induced morphological variation.

Domestic dogs and cats, 3 fox species, Dusicyon culpaeus (Molina,1782), D, griseus (Gray, 1837) and D. gymnocercus (Thomas, 1914), Geoffroy's cats, Felis geoffroyi (D'Orbigny and Gervais, 1843), and grisons, Galictus cuja (Molina, 1782) were fed larvae of Echinococcus granulosus (Batsch, 1786) from domestic sheep in Argentina. Dogs and the 3 species of foxes became infected. The susceptibility of D. culpaeus compared favorably to that of dogs. Most D. griseus and D. gymnocercus were less susceptible than dogs but gravid strobilae were recovered from some animals of both species. Eggs from strobilae in dogs and Dusicyon spp. were infective to CF1 mice. The mean strobilar length and the diameters of the rostellar pad and suckers of worms in foxes were significantly reduced when compared with previously reported data for strobilae from dogs. The potential significance of Dusicyon spp. as reservoir hosts of E. granulosus in Argentina is discussed as is the confusion surrounding the taxonomic identification of cestode infections previously reported from these wild carnivores. It is suggested that Echinococcus spp. reported from Argentine foxes are E. granulosus and that foxes become infected by killing and scavenging on sheep in localities where E. granulosus is endemic.

Animals

[The ecology of Echinococcus multilocularis (Leuckart, 1863) and E. granulosus (Batsch, 1786) on the Kamchatka Peninsula].

Some aspects of dwelling of Echinococcus multilocularis and E. granulosus populations in the Kamchatka Peninsula were considered. Transformation of their natural foci to natural synanthropic due to enrichment of mammalian fauna, increased human density and rural development was noticed. Main quantitative characteristics of infectivity of the worm definitive and intermediate hosts are presented using literature and the author's own data. A correlation was noted between host density and their infectivity with Echinococcus.

Animal Diseases

Histological preparation of the Echinococcus sp. tapeworms: staining affinity of the adult form.

Thirty specimens of Echinococcus sp. obtained by anti-helminthic treatment using arecoline hydrobromide from a dog from an Echinococcosis endemic region were fixed in 10% formalin. The material was processed histologically by a rapid manual method and stained using eleven different techniques. Green Trichrome staining as modified in the present study permitted the observation of large structures of Echinococcus sp. tapeworms.

Animals

[Construction of Echinococcus granulosus genomic library].

Using EMBL3 phage DNA as a vector, a perfect genomic library of Echinococcus granulosus from sheep from Xinjiang has been constructed, which contains 1.2 x 10(6) independent recombinants. The main procedure of construction comprised: 1. extraction of Echinococcus granulosus genomic DNA, 2. partial digestion of the extracted E. granulosus genomic DNA with restriction enzyme Sau3Al, 3. harvest of 15-23 kb DNA fragments by electric elution equipment, 4. preparation of EMBL3 phage vector DNA, 5. cleavage of EMBL3 vector DNA with two restriction enzymes Bam HI and Eco RI to remove the central fragment, 6. ligation of the harvested 15-23 kb E. granulosus genomic DNA and the cleavaged vector DNA with T4 DNA ligase, 7. the package reaction of ligated recombinant DNA in vitro with the package protein and with Q359 strain (Spi-) to screen recombinants, 8. identification of the genomic library, 9. hybridization in situ by pSM889 probe, obtaining 6 positive recombinant clones. It was estimated that the E. granulosus genome was about 1.5 x 10(8) bp. A perfect E.g. genomic library is expected to contain 4.6 x 10(4) independent recombinants at minimum. The constructed genomic library has enough independent recombinants (1.2 x 10(6)). This is the first time to establish an E. granulosus genomic library in China. Previous work showed that there existed differences in many aspects, including pathogenicity, among various isolates of E. granulosus from different hosts and areas. We plan to employ this library to screen the E. granulosus intraspecies DNA probe by hybridization in situ. This kind of probe is envisaged to be of advantage for epidemiological investigation of the hydatid disease in China. It also provides a base for researching E. granulosus at the molecular level.

Animals