The origin of a new focus of infection with Echinococcus granulosus in Tasmania.

Hydatid cysts were discovered in cattle on King Island, north of Tasmania, where Echinococcus granulosus was thought to have been eradicated. Using enzyme electrophoresis, isolates from King Island were compared genetically with isolates from Tasmania and the mainland of Australia. The genetic distinctness of the King Island isolates make it unlikely that they originated from a recent introduction from either Tasmania or mainland Australia. Alternative possibilities, that the infection resulted from a recent introduction from another source or from previously undetected persistence of E. granulosus on King Island, could not be distinguished from available data.     

Echinococcus granulosus: In vitro effectiveness of warm water on protoscolices

Hydatid disease is one of the most important helminthic diseases worldwide. Hydatid cysts may be found anywhere in the body. The most effective treatment of hydatid cyst is surgical operation. Spillage of live protoscolices during the operation is the major cause of recurrence. Instillation of scolicidal agent into hydatid cyst is the most commonly employed measure to prevent this complication. To date, many scolicidal agents have been used for inactivation of the hydatid cyst content, however, most common scolicidal agents may cause unacceptable side-effects, limiting their use. In this study the scolicidal effect of warm water (45, 50, 55, and 60 °C) at different exposure times (1, 2, 3, 4, 5, 6, 8, 10, 12, and 15 min) is investigated. Protoscolices were collected aseptically from sheep livers containing hydatid cyst. Viability of protoscolices was determined by 0.1% eosin staining. Even though the highest scolicidal activity of warm water at 45 °C was 40.4% at the end of 15 min, the best scolicidal effect (100%) of warm water at 50, 55, and 60 °C was obtained after 5, 2, and 1 min, respectively. The results of this in vitro study showed that warm water at 50–60 °C can be regarded as an effective scolicidal agent. Warm water is commonly available, easily prepared, and inexpensive. In vivo scolicidal activity of warm water and also the possible side effects need further investigation.     

Partial purification and kinetic properties of cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices

Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCl or barbital buffer. The κ_m values of oxaloacetate and NADH were 0.400 ± 0.018 and 0.410 ± 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65°C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a κ_i value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a κ_i value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated.     

Comparative chemical composition of protoscolices and hydatid cyst fluid of Echinococcus granulosus (Cestoda)

Hydatid cysts were analysed for electrolytes, nucleic acids, proteins, enzymes, nitrogenous waste products, carbohydrates and lipids. Substantial amounts of cations were detected. Potassium, magnesium and calcium were concentrated in protoscolices as compared to cyst fluid, but sodium levels were similar in both. Of the anions, chloride constituted the major bulk in the fluid anions, and was not found in protoscolices. In contrast, phosphate and bicarbonate had more significant values in the protoscolices. RNA and DNA which were found in substantial amounts in the protoscolices were nearly absent in the fluid ; so was ammonia. The reverse pattern of distribution was observed in urea, uric acid, creatinine and bilirubin. The proteins of cyst fluid were mainly albumin and globulin, the latter having always double the concentration of the former. Albumins and globulins also formed J of protoscolex protein. Among enzymes, LDH, phosphatases, GOT and GPT exhibited high activities. Cholesterol, cholesterol esters, mono-, di- and triglycerides, fatty acids and phospholipids were detected mainly in the protoscolices with phospholipids constituting the major portion. The carbohydrates of the protoscolices were glycogen, trehalose, glucose and alkali stable carbohydrates. Of particular interest was the detection of sucrose in both protoscolices and fluid.     

Geographic pattern of genetic variation in the fox tapeworm Echinococcus multilocularis

Intraspecific genetic variation of Echinococcus multilocularis, the etiologic agent of human alveolar echinococcosis, has been evaluated among 76 geographic isolates from Europe, Asia and North America by using sequence data of mitochondrial and nuclear DNA. Relatively low genetic variation was found only in the mitochondrial DNA sequence consisting of 3 protein-coding genes. Pairwise divergence among the resultant 18 haplotypes ranged from 0.03 to 1.91%. Phylogenetic trees and parsimony network of these haplotypes depicted a geographic division into European, Asian and North American clades, but 1 haplotype from Inner Mongolia was unrelated to other haplotypes. The coexistence of the Asian and North American haplotypes could be seen, particularly on the St. Lawrence Island in the Bering Sea. These data suggest an evolutionary scenario in which distinct parasite populations derived from glacial refugia have been maintained by indigenous host mammals. The nuclear DNA sequence for the immunodominant B cell epitope region of ezrin/radixin/moesin-like protein (elp) was extremely conservative, indicating that the elp antigen is available for immunodiagnosis in any endemic areas.     

Apparent dominance of the G1–G3 genetic cluster of Echinococcus granulosus strains in the central inland region of Portugal

Infection by the larval stage of the cestode Echinococcus granulosus causes a disease known as cystic echinococcosis or hydatidosis, which is one of the most widespread zoonotic infections of veterinary and medical importance. Numerous studies have shown that E. granulosus exists as a complex of strains differing in a wide variety of criteria. Ten distinct genotypes (G1–G10) have been identified with a potential impact on the pathology, epidemiology and the effect of the measures implemented for the control of hydatidosis. Our main objective was to carry out a preliminary analysis of the genotypes of E. granulosus circulating in the central inland region of Portugal.Parasite samples (hydatid cysts, n = 27) were isolated from the liver and lung of sheep and cattle. The DNA extracted from protoscoleces isolated from the fertile cysts served as a template for the PCR amplification of the part of the mitochondrial cytochrome c oxidase subunit 1 (cox1), ATP synthase F0 subunit 6 (atp6) as well as the large (rrnL/16 S) and small (rrnS/12 S) ribosomal RNA genes. Similarity searches with homologous sequences in the databanks indicated a very high similarity with references assigned to the G1, G3 and/or G1–G3 complex of Echinococcus strains. Phylogenetic analysis (Bayesian approach) supported these observations, and confirmed the assignment of all the analyzed sequences to the G1–G3 genetic cluster.     

Morphological variation of Echinococcus granulosus protoscoleces from hydatid cysts of human and various domestic animals in Jordan

, and 1988. Morphological variation of Echinococcus granulosus protoscoleces from hydatid cysts of human and various domestic animals in Jordan. International Journal for Parasitology 18: 1111–1114. Rostellar hook morphology of protoscoleces was employed to study the possible existence of Echinococcus granulosus strains in humans and various domestic animals in Jordan. A distinct form in the donkey was evident as the protoscoleces from this host did not share any characteristics with those from the other hosts examined. Sheep, goats and cattle appeared to be affected by another form since the protoscoleces from their hydatid cysts shared six out of nine characteristics studied. Protoscoleces of camel and human cysts shared seven out of nine characteristics studied and they were different in six characteristics from protoscoleces from other hosts. Differences observed among the three forms may reflect strain variation of E. granulosus in this country.     

Echinococcus granulosus myophilin-Relationship with protein homologues containing “Calponinmotifs”

Myophilin, a smooth-muscle protein of the tapeworm Echinococcus granulosus, was recently postulated to be a member of the calponin family of proteins. A detailed genetic analysis revealed that 17 proteins had significant homology with the amino-acid sequence of the N-terminal region of myophilin and/or possessed one or more “calponin-motifs”. Comparison of the amino-acid sequences of the N-terminus showed that the homologous proteins clustered into distinct groups based on the number of calponin-motifs. The calponin-motif of myophilin was genetically more similar to that present in the muscle protein mp20 of Drosophila melanogaster than to those in any other homologous proteins of vertebrates. The existence of a distinct motif which is “conserved” in other proteins across a range of species suggests an important functional role for the motif.     

Biochemical characterisation of Australian strains of Echinococcus granulosus by isoelectric focusing of soluble proteins

The existence of three distinct strains of E. granulosus in Australia has been previously demonstrated on the basis of several criteria. In the present study, numerous isolates of E. granulosus from domestic and wild animal populations in different geographical areas of Australia were subjected to detailed biochemical analysis using isoelectric focusing of soluble proteins. Three different populations were recognised which corresponded to the three strains described previously, thus confirming their genetic distinction. One strain is common to all domestic intermediate hosts on the Australian mainland. Evidence is presented that humans and macropod marsupials are also susceptible to infection with this strain and that it is similar to E. granulosus occurring in sheep in New Zealand and the United Kingdom. The other two strains are confined to macropod marsupials on the Australian mainland and sheep in Tasmania respectively.     

Formation of Echinococcus granulosus laminated membrane in a defined medium

Echinoccocus granulosus protoscoleces were digested from brood capsule material using artificial gastric fluid, and were cultured for 60 days in medium NCTC 135. Vesiculation occurred within 7 days, and the first laminated membranes were observed after 17 days of culture. Some contaminating sheep antigens appeared to be lost after 21 days.     

The Mongolian gerbil (Meriones unguiculatus) as a laboratory host for the cystic stage of Echinococcus granulosus of British horse origin

The Mongolian gerbil (Meriones unguiculatus) has been found to be susceptible to secondary equine hydatidosis and fertile cysts can be routinely produced with the subsequent serial passage through further generations. Mice, rats, rabbits, guinea-pigs, hamsters, cotton rats and voles proved to be refractory to infection. Since some of these hosts have been infected experimentally with horse material in other countries, by other workers, these results suggest that strain differences may exist between Echinococcus granulosus from the same host species subjected to geographical separation.     

Echinococcus granulosus: Protoscolicidal effect of high intensity focused ultrasound

High intensity focused ultrasound (HIFU) is a new non-invasive technique which can cause cell death and tissue necrosis by focusing high-energy ultrasonic waves on a single location. The aim of our work is to investigate the damaging effect of HIFU on Echinococcus granulosus protoscolices, as well as its inhibitory effect on growth of hydatid cysts derived from protoscolices. The damaging effect of HIFU on protoscolices was investigated by following parasite mortality after irradiation, while the inhibitory effect was investigated by infection experiments in vivo. The results demonstrated that HIFU was able to damage protoscolices and the protoscolicidal effect was dose-dependent and showed late-onset. The growth of protoscolices that survived the exposure to HIFU was obviously suppressed in vitro, and the mean weight of hydatid cysts resulting from such protoscolices in the experimental group was less than that in controls. Evidences including the protoscolicidal effect, fragmentized protoscolices and low post exposure temperatures, suggest that cavitation may contribute to the protoscolicidal effect of HIFU. In addition, the structure of the germinal membrane in cysts developing from the irradiated protoscolices was not as normal or intact as that from non-irradiated ones, and morphological changes related to degeneration were observed, suggesting that HIFU could prevent protoscolices from developing normal germinal membrane and consequently stop the proliferation of secondary hydatid cysts. HIFU demonstrated damaging effect on protoscolices, inhibited the growth of protoscolices in vitro and in vivo, and could be a possible therapeutic option for cystic echinococcosis.     

Echinococcus granulosus genotypes in livestock of Iran indicating high frequency of G1 genotype in camels

In this study, 112 Echinococcus granulosus isolates from different livestock of Iran were genotyped by PCR amplification of ribosomal DNA-internal transcribed spacer 1 (rDNA-ITS1) region followed by restriction fragment length polymorphism (RFLP) with the enzyme RsaI. The possibility of intra-genotype variation was also investigated using randomly amplified polymorphic DNA (RAPD) analysis. Isolates from sheep, goats, cattle and the majority of camels (12 of 18; 66.7%) were identified as the G1 genotype and a few camel isolates (6 of 18; 33.3%) belonged to the G6 genotype. Overall G1 and G6 genotypes were identified in 94.6% (106 of 112) and 5.3% (6 of 112) of all isolates, respectively. RAPD analysis based on 15 separate primers showed 7-14 bands of 200-3000 bp for strain G1. Considering each individual primer, no differences observed among isolates from different hosts and between livers and lungs. This study confirmed the existence of G1 and G6 genotypes in Iran. Moreover, G1 is much more prevalent even in camels, indicating the importance of sheep-dog cycle in public health. Studying intra-genotypic variation of E. granulosus warrants more research using other primers and methods.     

Assessment of the epidemiological status of Echinococcus multilocularis in foxes in France using ELISA coprotests on fox faeces collected in the field

The aim of this study was to estimate the relevance of Echinococcus multilocularis coproantigen detection in fox faeces collected in the field to identify different levels of endemicity for Echinococcus multilocularis on a large scale (n×10 km²). Six study sites were selected in a high endemicity area and two study sites in a low endemicity area in eastern France on the basis of landscape composition. Sampling was undertaken in the winters of 1996–97, 1997–98 and 1998–99. At each site, (i) necropsy and intestine examination was undertaken on a sample of shot foxes (total number of foxes, 222), and (ii) fox faeces were collected in the field along road verges, and scored for degradation status (total number of faeces, 625). Fox faeces were also sampled in a control area (n=30) in western France in the summer of 1998. Intestines were examined according to the sedimentation method. Echinococcus multilocularis coproantigens were detected by using two ELISA tests: EM-ELISA and EmA9-ELISA. The necropsy prevalence in high and low endemicity areas was 63.3% and 19.4%, respectively, and the distribution of adult worms in the fox population was highly overdispersed (75.5% of the total biomass was harboured by 11.6% of foxes). Using the two ELISA tests, there was no difference in the detection of E. multilocularis coproantigens in field faeces, regardless of the degradation status. The medians of EM- and EmA9-ELISA OD values of field faeces in high endemicity area were significantly higher than in low endemicity area (P<0.001 for both ELISA). The distribution of EM-ELISA OD values in low endemicity area was significantly higher (P=0.002) than in the control area. Moreover, for the two ELISA, the observed ELISA OD value distributions in high endemicity area, low endemicity area and control area seemed representative of the distribution of adult worms in fox populations. These results indicate that E. multilocularis coproantigen detection in field faeces could serve for large-scale surveillance, as an alternative to necropsy.     

Modelling the age variation of larval protoscoleces of Echinococcus granulosus in sheep

In autumn 2006, a study of the age-dynamics of Echinococcus granulosus cyst abundance was undertaken from an abattoir study of 1081 sheep slaughtered in Naryn Province in central Kyrgyzstan, an area endemic for echinococcosis. The results demonstrated approximately 64% of sheep were infected with the prevalence increasing markedly with age. The mean abundance was 3.8 cysts per sheep. From established models, an infection pressure of 1.33 cysts per year was estimated. In addition all cysts were recovered from infected sheep and the numbers of protoscoleces was evaluated in each cyst. A new model was developed that examined the variation in numbers of protoscoleces per infected sheep with age. This demonstrated that young sheep aged 1-2 years had very few protoscoleces, but there was a massive increase as the sheep aged. The best-fitting model assumed that the number of protoscoleces in a sheep was proportional to the volume of the cysts. In this model, the radius of the individual cyst increased linearly with the age of the cyst and hence the volume increased with the cube of the cyst age. This combined with the linear increase in numbers of cysts with age resulted in a massive accumulation of protoscoleces with the age of sheep. When the model was parameterised it demonstrated that 80% of protoscoleces were present in sheep aged 4 years and older and this represented just 28% sheep slaughtered. An average sheep at 6 or more years of age has an abundance of over 9700 protoscoleces, whilst in a young sheep of 1 year of age an average of just 16 protoscoleces could be found. The average for the sampled population across all ages was 1562 protoscoleces per sheep. The maximum number of protoscoleces in a single cyst was just 482 for sheep aged 1 year rising to 92,000 for sheep aged 6 years or older. The mean volume of cysts containing protoscoleces increased from approximately 0.7 ml at 1 year of age to 8.8 ml at 6 years of age. Cysts containing protoscoleces ranged from a diameter of 0.5-8 cm with a volume of fluid ranging from 0.2 to 50 ml. It is hypothesised that removal of old sheep through a culling programme could substantially improve the control of cystic echinococcosis.     

Coproantigen detection in dogs experimentally and naturally infected with Echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay

A sandwich ELISA for the detection of Echinococcus granulosus coproantigen in formalin and heat-treated faecal supernatants of dogs was developed. The assay used affinity-purified polyclonal antibodies obtained from rabbits hyperimmunised with E. granulosus excretory/secretory antigens and biotiaylated monoclonal antibody EmA9 produced against adult E. multilocularis somatic extract. The test was sensitive to 7 ng and 2.3 ng of E. granulosus protein and carbohydrate/ml of faecal supernatant, respectively. Thirteen helminth-free dogs were infected with different amounts of E. granulosus protoscoleces and the presence of coproantigen was monitored during the prepatent period until day 35 post-infection, when they were necropsied. Faecal antigen levels started to rise above the normal range between days 10 and 20 post-infection, and typically peaked at the end of the experiment. All the dogs, bearing from 3 to 67 700 worms, showed positive values in the ELISA during the prepatent period. One dog experimentally infected with Taenia hydatigena metacestode and harbouring three worms, tested positive only after the prepatent period at day 52. The test was applied to 98 stray dogs. The ELISA detected all of four dogs naturally infected with E. granulosus, two dogs with patent infections of T. hydatigena and two dogs with no cestode infections, sbowing a sensitivity of 100% and a specificity of 96%.     

A new fungal genus, Teracosphaeria, with a phialophora-like anamorph (Sordariomycetes, Ascomycota)

The new genus and species Teracosphaeria petroica is described for a perithecial ascomycete and its anamorph occurring on decayed wood collected in New Zealand. The fungus produces immersed, non-stromatic ceratosphaeria-like perithecia in nature, with hyaline, septate ascospores produced in unitunicate, non-amyloid asci. The anamorph produced in vitro is phialophora-like with lightly pigmented phialides terminating in flaring, deep collarettes that are often noticeably brown with conspicuous periclinal thickening. Phylogenetic analysis of LSU rDNA sequence data indicates that this fungus is distinct from morphologically similar fungi classified in the Chaetosphaeriales, the Trichosphaeriales or the Magnaporthaceae. It forms a monophyletic group with recently described, chaetosphaeria-like ascomycetes, such as the pyrenomycete genus Mirannulata, and shows affinity with the anamorphic species Dictyochaeta cylindrospora. The usefulness of describing anamorph genera for morphologically reduced anamorphs, when anamorph characteristics are actually part of the holomorph diagnosis, is discussed. An apparently contradictory example of the so-called Cordana and Pseudobotrytis anamorphs of Porosphaerella spp. is also discussed.     

Corylomyces: a new genus of Sordariales from plant debris in France

The new genus Corylomyces, isolated from the surface of a hazelnut (Corylus avellana) in the French Pyrenees, is described, illustrated and compared with morphologically similar taxa. It is characterised by tomentose, ostiolate ascomata possessing long necks composed of erect to sinuose hairs, and one- or two-celled, opaque, lunate to reniform ascospores. Analyses of the SSU and LSU fragments rDNA gene sequences support its placement in the Lasiosphaeriaceae (Sordariales).