Biochemical profiles of hydatid cyst fluids of Echinococcus granulosus of human and animal origin in Libya.

A comparative study on the biochemical parameters in hydatid cyst fluids of sheep, goats, camels, cattle and human cystic forms of Echinococcus granulosus has been made in Libya. Quantitative variations in the levels of sodium, potassium, calcium, cholesterol, glucose, urea, creatinine and gamma glutamyl transpeptidase (gamma GT) were found in the cystic fluids of different host origins although these differences were statistically insignificant compared with the hydatid fluids of sheep. However, the concentration of triglycerides and proteins were significantly elevated in the cyst fluids of sheep compared with the other fluids studied. Similarities in the biochemical composition of different hydatid cyst fluids suggest the existence of sheep strains of E. granulosus in human and other domestic animal intermediate hosts in Libya.     

A reconsideration of the Echinococcus granulosus strain situation in Australia following RFLP analysis of cystic material.

Restriction fragment length polymorphism (RFLP) analysis, previously shown to be a relatively simple and reproducible method for distinguishing discrete strains of E. granulosus, could not discriminate between E. granulosus originating from central Queensland macropod marsupials, Australian mainland sheep or United Kingdom sheep. Furthermore, sheep cyst material from Tasmania and the Australian mainland was indistinguishable using this approach. As a result of this DNA analysis, the existence of three distinct strains of E. granulosus in Australia, previously described on the basis of morphological, biochemical and developmental differences, may require re-evaluation.     

Molecular characterization of Echinococcus granulosus in Tunisia and Mauritania by mitochondrial rrnS gene sequencing

Cystic hydatid disease is a zoonotic parasitic disease caused by the cestode Echinococcus granulosus and represents a major public health problem in many countries around the world, including North Africa. E. granulosus exists as a series of genetic variants or strains which differ in a wide variety of criteria that impact on the epidemiology, pathology and control of cystic hydatid disease. Nucleotide sequencing of the mitochondrial rrnS gene was here used to characterize 38 E. granulosus isolates collected from different regions and hosts in Tunisia and Mauritania. The results obtained reveal a significant genetic differentiation between E. granulosus hydatid cysts identified as belonging to the G1 genotype and to the G6/G7 cluster using the rrnS gene as marker, and indicate the circulation of the common sheep strain (G1) in all host species from Tunisia and the camel/pig strain cluster (G6/G7) in camel from Mauritania. Other investigations, using this method, are necessary for further genetic analysis of a wider range of isolates from different host species in order to more fully understand the genetic structure of E. granulosus populations and their transmission dynamics in this and neighbouring African countries.     

Experimental infection of the baboon (Papio cynocephalus) with Echinococcus granulosus of camel, cattle, sheep and goat origin from Kenya

In different areas of the world, strains of Echinococcus granulosus have been described which appear to vary in their infectivity, and laboratory primates have been used as indicators of their infectivity to man. This phenomenon was evaluated in Kenya for hydatid material of human, camel, cattle, sheep and goat origin. Viable eggs, produced by experimental infections in dogs with larval material from all the above intermediate hosts, were fed separately to four baboons (Papio cynocephalus) in each case. Baboons were autopsied between 373 and 501 days following infection and the liver, lungs, heart, spleen and kidneys were thoroughly inspected. Hydatid cysts were recovered from two baboons in each of the camel, sheep and goat groups, three baboons in the cattle group and none of the baboons in the human group. Fertile cysts were found in the cattle and goat groups and it is suggested that the baboon could be used as an experimental model for this parasite.     

A PCR system for detection of species and genotypes of the Echinococcus granulosus-complex, with reference to the epidemiological situation in eastern Africa

We describe the development of a specific and sensitive PCR/semi-nested PCR system for the rapid diagnosis of Echinococcus granulosus genotype G1, E. granulosus genotype G6/7, and Echinococcus ortleppi (G5). Diagnosis of G1 and the group G5/6/7 is performed by a simple PCR, while discrimination between E. ortleppi (G5) and G6/7 involves a subsequent semi-nested PCR step. The target sequence for amplification is part of the mitochondrial 12S rRNA gene. Specificity of the PCRs was 100% when evaluated with isolates of 16 species of cestodes, including Echinococcus multilocularis, Echinococcus equinus, E. ortleppi and three strains of E. granulosus (G1, G6 and G7). Sensitivity threshold was 0.25pg of DNA. This new approach was compared with published protocols of restriction fragment length polymorphism-PCR and sequencing of mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 genes using Echinococcus isolates of human, sheep, goat, camel, cattle and pig origin from Kenya and Sudan. Additionally, two internal DNA probes were developed, one hybridising only with G1, the other with G5, G6 and G7 amplification products. Preliminary epidemiological results obtained with this PCR approach include the detection of a camel strain (G6) infection for the first time in a human patient from eastern Africa, and the first reports of E. ortleppi (G5) in livestock from Kenya and the Sudan.     

Genetic characterization of Echinococcus granulosus in camels, cattle and sheep from the south-east of Iran indicates the presence of the G3 genotype

Echinococcus granulosus, the aetiologic agent of cystic echinococcosis (CE), is one of the most important zoonotic helminthes worldwide. Isolates of the parasite show considerable genetic variation in different intermediate hosts. Several genotypes and species are described in different eco-epidemiological settings. This study investigated E. granulosus genotypes existing in livestock and humans from the province of Kerman, located in south-eastern Iran, using sequencing data of cox1 and nad1 mitochondrial genes. Fifty-eight E. granulosus isolates, including 35 from sheep, 11 from cattle, 9 from camels and 3 from goats, were collected from slaughterhouses throughout Kerman. One human isolate was obtained from a surgical case of CE. Mitochondrial cox1 and nad1 regions were amplified using polymerase chain reaction (PCR) and 38 isolates were sequenced. Genotypes G1 (73.7%), G3 (13.2%) and G6 (13.1%) were identified from the isolates. G1 was the most common genotype from sheep (86.7%), cattle (80%), camels (44.4%) and goats (100%). Sheep, cattle and camels were also found to be infected with the G3 genotype (buffalo strain). The human isolate was identified as the G6 genotype. Results showed that the G3 genotype occurred in different animal hosts in addition to G1 and G6 genotypes.     

The epidemiology of hydatidosis with special reference to the Mediterranean area.

In the Mediterranean area 2 subspecies of Echinococcus granulosus exist: E. g. equinus and E. g. granulosus. The latter is divided into sheep, cattle, pig and camel strains, the adult forms of which mainly occur in farm-dogs but sometimes in wild canidae. Some of these strains can infect man: namely, the sheep and the pig strain. The infectivity of the cattle strains for man is not certain. The epidemiological cycle is mainly a rural one, but it can become sylvatic or even urban. Thus, the epidemiological features of hydatidosis in the Mediterranean area are not basically different from the general epidemiology of the disease. But extensive rearing of sheep, combined with the carelessness and ignorance of people and with some particular habits, favours the maintenance of the infection.     

Morphological characteristics of Echinococcus granulosus derived from buffalo in Iran

Cystic echinococcosis is a significant parasitic disease in Iran, where a variety of animals act as intermediate hosts. In this study, 25 isolates of Echinococcus granulosus obtained from water buffalo from various parts of Iran were characterized on the basis of the morphology of the metacestode and the adult worm. The characteristics of protoscoleces from the different studied areas were nearly similar. They showed 2 rows of alternating large and small hooks and their shapes were smooth in outline. In contrast to the protoscoleces, the adult rostellar hooks showed a rough outline. The results showed that the total length, the blade lengths of the large and small hooks and the number of hooks are almost similar to those isolated from sheep but significantly different from those isolated from camels. The growth rates of adult E. granulosus (total worm length, segmentation and maturation) of buffalo origin, at 35 and 41 days post-infection of dogs, were nearly comparable to the common sheep strain. The form of the strobila and the morphology of the reproductive system were also similar to those of sheep origin. This suggests that the common sheep strain (G1) of E. granulosus may also use buffaloes as its intermediate host.     

The action of albendazole on hydatid cysts in sheep experimentally infected with eggs of Echinococcus granulosus

According to the Brazilian Agricultural Ministry, the prevalence of cystic echinococcosis in the state of Rio Grande do Sul, Brazil has decreased between 1990 and 1998, possibly due to the common use of albendazole to control parasites in sheep. Therefore, albendazole (ABZ) action was tested on hydatid cysts of sheep experimentally infected with eggs of Echinococcus granulosus in Uruguaiana County (RS) with the aim of evaluating the observed changes in the parasite's prevalence. The sheep were divided into three groups of 15 animals each and treated as follows. Group 01 was a control (without treatment), groups 02 and 03 received 7.5 mg/kg and 15 mg/kg, respectively, of ABZ orally for 12 months, at intervals between 36 and 45 days. At the end of the studied period the animals were necropsied and the viability of the hydatid cysts was evaluated. Lung and hepatic cysts of sheep treated with ABZ were reduced in size. Degeneration and evident calcification were seen in 87.3% (group 02) and 75.7% (group 03), which were significantly different (P < 0.01) from the 17.3% demonstrated in the control group. It could be concluded that ABZ used early and over long periods, even in small doses, is capable of promoting lesions that devitalize the cysts. This leads to a decrease in fertility of E. granulosus in sheep and could be the reason why the prevalence of cystic echinococcosis has decreased in recent years.     

Molecular identification of unilocular hydatid cysts from domestic ungulates in Ethiopia: implications for human infections

To identify the etiologic agents of cystic echinococcosis in Ethiopia, unilocular hydatid cysts were collected from 11 sheep, 16 cattle and 16 camels slaughtered in abattoirs of Aweday, Jijiga, Haramaya and Addis Ababa during June 2010 to February 2011. A PCR-based DNA sequencing of the mitochondrial cytochrome oxidase c subunit 1 gene (cox1) was conducted for 40 cysts. The majority of cysts (87.5%) were identified as Echinococcus granulosus sensu stricto and the rest as Echinococcus canadensis. The fertile cysts of E. granulosus s.s. were found only from sheep, although it occurred in all the host species. The predominance of E. granulosus s.s. has important implications for public health since this species is the most typical causative agent of human cystic echinococcosis worldwide. The major cox1 haplotype of E. granulosus s.s. detected in Ethiopia was the same as that has been reported to be most common in Peru and China. However, a few cox1 haplotypes unique to Ethiopia were found in both of the two Echinococcus species. The present regional data would serve as baseline information in determining the local transmission patterns and in designing appropriate control strategies.     

Echinococcus granulosus genotypes circulating in alpacas (Lama pacos) and pigs (Sus scrofa) from an endemic region in Peru

The identification of the genotypes of Echinococcus granulosus present in livestock and wild animals within regions endemic for cystic echinococcosis (CE) is epidemiologically important. Individual strains display different biological characteristics that contribute to outbreaks of CE and that must be taken into account in the design of intervention programs. In this study, samples of hydatid cysts due to E. granulosus were collected from alpacas (4) in Puno and pigs (8) in Ayacucho in Peru, an endemic region for CE. Polymerase chain reaction amplification and DNA sequencing of specific regions of the mitochondrial cytochrome C oxidase subunit 1 and NADH dehydrogenase subunit 1 genes confirmed the presence of a strain common to sheep, the G1 genotype, in alpacas. Two different strains of E. granulosus were identified in pigs: the G1 and the G7 genotypes. This is the first report of the G1 genotype of E. granulosus in alpacas in endemic regions of CE in Peru.     

Echinococcus granulosus sensu lato genotypes infecting humans – review of current knowledge

Genetic variability in the species group Echinococcus granulosus sensu lato is well recognised as affecting intermediate host susceptibility and other biological features of the parasites. Molecular methods have allowed discrimination of different genotypes (G1–10 and the ‘lion strain’), some of which are now considered separate species. An accumulation of genotypic analyses undertaken on parasite isolates from human cases of cystic echinococcosis provides the basis upon which an assessment is made here of the relative contribution of the different genotypes to human disease. The allocation of samples to G-numbers becomes increasingly difficult, because much more variability than previously recognised exists in the genotypic clusters G1–3 (=E. granulosus sensu stricto) and G6–10 (Echinococcus canadensis). To accommodate the heterogeneous criteria used for genotyping in the literature, we restrict ourselves to differentiate between E. granulosus sensu stricto (G1–3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and E. canadensis (G6–7, G8, G10). The genotype G1 is responsible for the great majority of human cystic echinococcosis worldwide (88.44%), has the most cosmopolitan distribution and is often associated with transmission via sheep as intermediate hosts. The closely related genotypes G6 and G7 cause a significant number of human infections (11.07%). The genotype G6 was found to be responsible for 7.34% of infections worldwide. This strain is known from Africa and Asia, where it is transmitted mainly by camels (and goats), and South America, where it appears to be mainly transmitted by goats. The G7 genotype has been responsible for 3.73% of human cases of cystic echinococcosis in eastern European countries, where the parasite is transmitted by pigs. Some of the samples (11) could not be identified with a single specific genotype belonging to E. canadensis (G6/10). Rare cases of human cystic echinococcosis have been identified as having been caused by the G5, G8 and G10 genotypes. No cases of human infection with G4 have been described. Biological differences between the species and genotypes have potential to affect the transmission dynamics of the parasite, requiring modification of methods used in disease control initiatives. Recent investigations have revealed that the protective vaccine antigen (EG95), developed for the G1 genotype, is immunologically different in the G6 genotype. Further research will be required to determine whether the current EG95 vaccine would be effective against the G6 or G7 genotypes, or whether it will be necessary, and possible, to develop genotype-specific vaccines.     

Detection of genetic polymorphism among and within Echinococcus granulosus strains by heteroduplex analysis of a microsatellite from the U1 snRNA genes

Polymerase chain reaction of a pentanucleotide microsatellite in the U1 snRNA gene complex generated a multiple band pattern due to the priming of paralogous sequences. Denaturation and slow renaturation of polymerase chain reaction products allow the formation of heteroduplex DNA that can be detected by its differential mobility in polyacrylamide gel electrophoresis. Heteroduplex analysis was used to determine if the U1 snRNA microsatellite could be a useful genetic marker in Echinococcus granulosus. A U1 snRNA microsatellite fragment from E. granulosus was isolated and characterized by Southern blot and sequencing. Four E. granulosus strains were analyzed: sheep, Tasmanian sheep, cattle, and camel strains. The former two showed polymorphism and shared three of the six patterns found for sheep strain. The cattle strain displayed two patterns, and the camel strain was monomorphic. The electrophoretic profiles were used for statistical analysis in order to determine genetic distance and the relationship among strains. Heteroduplex analysis can be helpful in genotyping E. granulosus strains and is useful in detecting polymorphism within strains.     

Identification of Echinococcus granulosus microRNAs and their expression in different life cycle stages and parasite genotypes

The aetiological agent of cystic hydatid disease, the platyhelminth parasite Echinococcus granulosus, undergoes a series of metamorphic events during its complex life cycle. One of its developmental stages, the protoscolex, shows a remarkable degree of heterogeneous morphogenesis, being able to develop either into the vesicular or strobilar direction. Another level of complexity is added by the existence of genotypes or strains that differ in the range of intermediate hosts where they can develop and form fertile cysts. These features make E. granulosus an interesting model for developmental studies. Hence, we focused on the study of the regulation of gene expression by microRNAs (miRNAs), one of the key mechanisms that control development in metazoans and plants and which has not been analysed in E. granulosus yet. In this study, we cloned 38 distinct miRNAs, including four candidate new miRNAs that seem to be specific to Echinococcus spp. Thirty-four cloned sequences were orthologous to miRNAs already described in other organisms and were grouped in 16 metazoan miRNA families, some of them known for their role in the development of other organisms. The expression of some of the cloned miRNAs differs according to the parasite life cycle stage analysed, showing differential developmental expression. We did not detect differences in the expression of the analysed miRNAs between protoscoleces of two parasite genotypes. This work sets the scene for the study of gene regulation mediated by miRNAs in E. granulosus and provides a new approach to study the molecules involved in its developmental plasticity and intermediate host specificity. Understanding the developmental processes of E. granulosus may help to find new strategies for the control of cystic hydatid disease, caused by the metacestode stage of the parasite.     

Strain characterization of Echinococcus granulosus protoscoleces of cattle origin using the in vitro vesicular development

The aim of this work was to characterize the strain of protoscoleces of E. granulosus of cattle origin using the in vitro vesicular development. The in vitro development of these samples was compared to samples of sheep origin determined previously by genetic analyses as common sheep strain (G1). There were similarities between sheep and cattle samples not only in the time of microcysts formation, but also in the development process. Vesiculated protoscoleces and protoscoleces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoleces. In the sheep samples, microcysts were observed between 19 and 20 days. In the cattle samples, microcysts appeared between 20 and 23 days. The coincidence between the development times and physiological characteristics found in the present study may indicate that the parasites from cattle and sheep were of the same strain.     

Development of a new PCR protocol for the detection of species and genotypes (strains) of Echinococcus in formalin-fixed, paraffin-embedded tissues

The aim of our study was to establish a new PCR protocol for the detection and discrimination of Echinococcus granulosus complex on one hand and Echinococcus multilocularis in formalin-fixed, paraffin-embedded tissues (FFPTs) on the other. The target sequences for all PCRs are located on a 471bp segment of the mitochondrial ND1 gene, the fragment sizes of the amplification products are 295bp (for the sheep strain of E. granulosus), 204bp (for the pig strain of E. granulosus) and 252bp (for E. multilocularis), respectively. In total, 80 FFPTs from patients with histologically confirmed echinococcosis (76 with E. granulosus and four with E. multilocularis) operated on in Austrian hospitals between 1978 and 2005 were examined. In 68 (85%) samples, we were able to detect specific DNA fragments with our newly established PCR protocols. Thirty-eight (47.5%) of 80 clinical samples were identified as the G1 strain, 26 (32.5%) as the G5, 6 or 7 strains and four (5%) as E. multilocularis. The specificity of all three PCRs was 100%; for the discrimination between G6 and G7 strains, sequencing of an additional 234bp PCR fragment was necessary and showed that three out of 26 G5, 6 or 7 PCR-positive patients were infected with E. granulosus genotype G6 (the camel strain).     

Differential Exoproteome Analysis of Two Corynebacterium pseudotuberculosis Biovar Ovis Strains Isolated from Goat (1002) and Sheep (C231)

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis a chronic infectious disease affecting small ruminants. The 2D-DIGE technique was used to compare the exoproteomes of two C. pseudotuberculosis biovar ovis strains isolated from goat (strain 1002) and sheep (strain C231). Seventeen proteins differentially produced were identified here. Nine proteins appeared over-produced in the exoproteome of 1002 goat strain and 8 in that of C231 sheep strain. These proteins were related to various biological functions, such as the cell envelope, respiratory metabolism and proteolysis. This proteomic analysis revealed strain-specific exoproteins although each of the corresponding genes was found in both strain genomes. Such differential expression pattern may reflect inter-strain differences in adaptation to a specific host, in pathogenicity and or in antigenicity of this pathogenic bacterium.     

Identification of membrane-bound and secreted proteins from Echinococcus granulosus by signal sequence trap

The signal sequence trap technique was applied to identify genes coding for secreted and membrane bound proteins from Echinococcus granulosus, the etiologic agent of cystic hydatid disease. An E. granulosus protoscolex cDNA library was constructed in the AP-PST vector such that randomly primed cDNAs were fused with a placental alkaline phosphatase reporter gene lacking its endogenous signal peptide. E. granulosus cDNAs encoding a functional signal peptide were selected by their ability to rescue secretion of alkaline phosphatase by COS-7 cells that had been transfected with the cDNA library. Eighteen positive clones were identified and sequenced. Their deduced amino acid sequences showed significant similarity with amino acid transporters, Krebs cycle intermediates transporters, presenilins and vacuolar protein sorter proteins. Other cDNAs encoded secreted proteins without homologues. Three sequences were transcribed antisense to E. granulosus expressed sequence tags. All the mRNAs were expressed in protoscoleces and adult worms, but some of them were not found in oncospheres. The putative E. granulosus secreted and membrane bound proteins identified are likely to play important roles in the metabolism, development and survival in the host and represent potential targets for diagnosis, drugs and vaccines against E. granulosus.     

A comparative study of Echinococcus granulosus from human and animal hosts in Kenya using isoelectric focusing and isoenzyme analysis

Macpherson C. N. L. and Mcmanus D. P. 1982). A comparative study of Echinococcus granulosus from human and animal hosts in Kenya using isoelectric focusing and isoenzyme analysis. International Journal for Parasitology12: 515–521. The soluble enzyme extracts from protoscoleces obtained from hydatid cysts of human, camel, cattle, sheep and goat origin were compared on the basis of their isoenzyme patterns for GPI and PGM using isoelectric focusing. Consistent GPI and PGM isoenzyme patterns were obtained for larvae of human, camel and sheep material. Cattle material varied occasionally in having an additional cathodic band in some of the GPI patterns. Two distinct isoenzyme patterns were evident in the goat material for both enzymes. The more common goat patterns were similar to those of human, cattle and sheep (Kenya, U.K. and Argentina) material, which were similar to each other. The rare goat patterns were similar to those obtained for camel material. Cyst location in the various intermediate hosts had no effect on the zymograms obtained. Additionally, no alteration in the major banding patterns was observed between the larvae and homologous adults produced by experimental infections. Of 26 naturally infected dogs, 19 produced adult GPI zymograms resembling human/ sheep/goat (common form) experimental infection patterns, three were similar to experimental cattle infections and four had camel/goat (rare form) patterns.     

Echinococcus granulosus strain differentiation in Iran based on sequence heterogeneity in the mitochondrial 12S rRNA gene

Parasite strain characterization is essential for the establishment of a prevention and control strategy in any endemic area. The aim of this study was to characterize different Echinococcus granulosus isolates from Iran by using DNA sequences of the mitochondrial 12S rRNA gene. Thirty livers and lungs of cattle, sheep and goats naturally infected with E. granulosus were collected from abattoirs in northern and western Iran between June and October 2007. These samples yielded 18 fertile cysts which we used for the genetic work. We designed and tested two new primer pairs which specifically amplify portions of the mitochondrial 12S rRNA gene of the two strains (G1 and G6) of E. granulosus known to occur in Iran. One primer pair amplified a fragment of 259 base pairs (bp) from only the G1 strain. The second pair amplified a fragment of 676 bp from the G6 strain. The G1 genotype was identified in all fertile cyst samples, in agreement with previous studies in Iran. Ten of our samples and a single reference sample of the G6 strain were sequenced and compared with the G1 and G6 sequences deposited in GenBank.