Detection ofEchinococcus coproantigens by enzyme-linked immunosorbent assay in dogs,dingoes and foxes

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection ofEchinococcus coproantigens in fecal samples from dogs, dingoes or foxes infected with eitherE. granulosus orE. 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 >1,000E. multilocularis worms and 13 of 15 (87%) samples from dogs or dingoes containing >200E. granulosus worms were ELISA-positive, whereas 34 of 46 samples from foxes harboring <1,000E. multilocularis and 9 of 10 samples from dogs or dingoes bearing <200E. granulosus tested negative. Experimental prepatent infections of dogs withE. granulosus revealed positive ELISA reactions within the prepatent period (10–20 days post-infection) for six animals bearing >1,000E. granulosus each; a low worm burden (<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 withE. multilocularis tested positive in the coproantigen ELISA as early as on day 5 post-infection.Dedicated to Prof. K.T.F. Friedhoff on the occasion of his 60th birthday     

Detection of Echinococcus multilocularis in the Definitive Host: Coprodiagnosis by PCR as an Alternative to Necropsy

Recently, extensions of the range of Echinococcus multilocularis in Europe and North America and drastic increases in fox populations in Europe put an increasing proportion of the human population at risk of alveolar echinococcosis. To obtain data on the local infection pressure, studies of the prevalence of the parasite in the animals that transmit the parasite, foxes, dogs, and cats, are urgently required. Such investigations, however, have been hampered by the need for necropsy of the host animal to specifically diagnose infection with the parasite. In this study, a nested PCR and an improved method for DNA extraction were developed to allow the sensitive and specific diagnosis of E. multilocularis infections directly from diluted fecal samples from foxes. The target sequence for amplification is part of the E. multilocularis mitochondrial 12S rRNA gene. The specificity of the method was 100% when it was tested against 18 isolates (metacestodes and adult worms) of 11 cestode species, including E. granulosus. The sensitivity of the method was evaluated by adding egg suspensions and individual eggs to samples of diluted feces from uninfected foxes. The presence of one egg was sufficient to give a specific signal. To confirm the PCR results, an internal probe which hybridized only with E. multilocularis amplification products but not with the DNA of other cestodes was constructed. In order to investigate the applicability of this method for epidemiological studies, 250 wild foxes from a area in southern Germany where echinococcosis is highly endemic were examined by both necropsy and PCR of rectal contents. The sensitivity correlated with the parasites’ number and stage of maturity. It ranged from 100% (>1,000 gravid worms) to 70% (<10 nongravid worms). On the basis of positive PCR results for 165 foxes, the sensitivity of the traditional and widely used necropsy method was found to be not higher than 76%. We therefore present this PCR system as an alternative method for the routine diagnosis of E. multilocularis in carnivores.     

Polymerase chain reaction for detection of patent infections of<Emphasis Type="Italic"> Echinococcus granulosus</Emphasis> (“sheep strain”) in naturally infected dogs

Polymerase chain reaction (PCR) for the identification of eggs of the tapeworm Echinococcus granulosus (sheep strain) was evaluated with primers derived from mitochondrial sequences. Specificity of these primers was confirmed by investigating DNA of other strains of E. granulosus and of 14 helminth species which inhabit the intestines of dogs. This PCR assay was used to investigate 131 purged dogs from Kazakhstan. Eighteen dogs harboured Echinococcus worms, ten of them in mixed infections with Taenia spp. Coproantigen detection was positive in 15 and taeniid eggs could be recovered from 13 of these specimens. Eight of the egg-containing samples were positive in the PCR for E. granulosus and four in a Echinococcus multilocularis -specific PCR revealing one mixed infection. Egg-containing faeces from two dogs harbouring both Taenia spp. and Echinococcus spp. were negative in both PCRs. The combination of egg isolation and PCR will also be of value in epidemiological studies when investigating environmental samples.     

Echinococcus granulosus in Dogs: Natural and Acquired Immunity Factors Interfering with Development During the Rapid Growth Phase of

Twenty-five dogs were used in a vaccination trial against infections of Echinococcus granulosus. Nine of these acted as controls and sixteen were vaccinated with freeze-dried preparations from either tapeworm tissue or scolices of E. granulosus. All were challenged with an aliquot of living scolices and examined at periods up to 49 days after challenge.

The results indicate that 0.1 g. of either crude antigen complex did not prevent some worms developing from a subsequent challenge with about 50,000 scolices. However, the number of segments and lengths of the subterminal and terminal segments of E. granulosus were almost always less than those found in controls. A phase of rapid growth of the terminal and subterminal segments was observed in control dogs. This was absent in practically all worms in vaccinated dogs and appears to be essential for egg production.

     

Detection of<Emphasis Type="Italic"> Echinococcus multilocularis</Emphasis> antigens in faeces by ELISA

Faecal samples deriving from 391 animals belonging to nine species (polecats, badgers, martens, weasels, rats, dogs, cats, red foxes, raccoon-dogs) were examined by capture ELISA for the presence of the Echinococcus multilocularis coproantigen. The main claim of our studies is the reliable detection of E. multilocularis coproantigens, mainly in the faeces of foxes, dogs and cats. For the first time in coproantigen detection we used a double-sandwich ELISA. The main advantage of this method is the higher specificity and better differentiation of positive and negative faecal samples, in comparison with sandwich ELISA. The overall specificity of double-sandwich ELISA was 95.1% with only 16 of 327 E. multilocularis-free animals giving false-positive results. The E. multilocularis coproantigen was detected by double-sandwich ELISA in 37.5% of examined red foxes and in 8.0% of examined raccoon-dogs, compared with a prevalence of just 29.8% in red foxes and 8.0% in raccoon-dogs, as determined by parasitological techniques.     

Efficacy of Emodepside plus Praziquantel Tablets (Profender? Tablets for Dogs) against Mature and Immature Cestode Infections in Dogs

The efficacy of a novel flavoured tablet formulation of emodepside plus praziquantel (Profender? tablets for dogs) against intestinal cestodes was investigated in four randomised, blinded placebocontrolled dose confirmation studies in dogs experimentally infected with Echinococcus granulosus or E. multilocularis and in dogs naturally infected with Dipylidium caninum or Taenia spp. The tablets were used at the minimum recommended dose of 1 mg emodepside and 5 mg praziquantel per kg body weight. The studies demonstrated 100% efficacy against mature and immature E. granulosus and E. multilocularis and mature Taenia spp. and D. caninum. Additionally, one of the studies demonstrated non-interference of emodepside with the efficacy of praziquantel against D. caninum. No side effects of the treatment were observed. It is concluded that emodepside plus praziquantel tablets are safe and effective against mature and immature stages of E. granulosus and E. multilocularis and mature stages of Taenia spp. and D. caninum.     

Cystic Echinococcosis

Echinococcosis is one of the 17 neglected tropical diseases (NTDs) recognized by the World Health Organization. The two major species of medical importance are Echinococcus granulosus and Echinococcus multilocularis. E. granulosus affects over 1 million people and is responsible for over $3 billion in expenses every year. In this minireview, we discuss aspects of the epidemiology, clinical manifestations, and diagnosis of cystic echinococcosis or cystic hydatid disease caused by E. granulosus.     

Echinococcus multilocularis infections in dogs from urban and peri-urban areas in France

Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe zoonotic disease. It is maintained through a sylvatic life cycle based on predator–prey interactions mainly between foxes and rodents. Dogs are also good definitive hosts; and due to their close proximity to humans, they may represent a major risk factor for the occurrence of human cases. In two medium-sized cities of Eastern France (Annemasse and Pontarlier), located in highly endemic areas, 817 dog feces samples were collected and analyzed by a flotation technique followed by a multiplex PCR assay. For the first time in France, we assessed the presence of E. multilocularis DNA in four dog feces samples, in which it represents an estimated prevalence of 0.5 % (95% CI; 0.1 % <> 1.3 %). Eight other samples presented taeniid infections from three different species (Taenia crassiceps, Taenia serialis, and Taenia polyacantha). When considering both E. multilocularis and Taenia sensu lato, prevalence rose to 0.6 % in Annemasse and 2.6 % in Pontarlier. In this highly endemic context, proper application of the usual deworming recommendations (70 % of the dogs were treated twice a year or more) failed to prevent dog infection, particularly for hunting dogs. Our results stressed the need to adapt treatment to the environmental context and to the specific activity of dogs. Further epidemiological surveys in domestic dogs and cats using this coprological approach are still needed to obtain a better overview of infection and the associated zoonotic risk.     

Molecular characterization of Echinococcus granulosus in a hyperendemic European focus,the Republic of Moldova

Cystic echinococcosis is a zoonosis caused by the tapeworm Echinococcus granulosus sensu lato. The lifecycle of the parasite is mainly domestic, requiring dogs as definitive hosts and livestock species as intermediate hosts. Although human cystic echinococcosis is a high public health priority in the Republic of Moldova, the rare animal data available concerns only infection in cattle. A preliminary slaughterhouse survey was conducted to assess prevalence and perform the first molecular characterization of E. granulosus sensu lato in sheep and cattle. For the survey, 40 sheep and 19 cattle were inspected. Very high prevalence in sheep (82.5 %) and in cattle (78.9 %) was found. Molecular analyses identified genotypes G1 and G3 of E. granulosus sensu stricto in all the liver and lung samples. Based on the concatenated sequences of cox1?+?nad3 (701 bp), 23 different haplotypes were obtained. Mixed infections by different haplotypes/genotypes were frequently identified in both sheep and cattle. The relatively high (20.0 %) cyst fertility observed in cattle argues for the potential contribution of cattle to the lifecycle of E. granulosus sensu stricto, unlike previous observations in Europe. The hyperendemic situation of Moldova can be explained by a high majority of animals slaughtered at home usually without veterinary inspection. Further extensive slaughterhouse surveys with molecular identification also involving pigs and goats are needed to obtain a better overview of the epidemiological situation of E. granulosus sensu lato in this hyperendemic focus in the Republic of Moldova.     

Serological differentiation betweenEchinococcus granulosus andE. multilocularis infections in man

An enzyme-linked immunosorbent assay (ELISA) was adapted for the serological differential diagnosis of cystic or alveolar echinococcosis in man caused byEchinococcus granulosus orE. multilocularis respectively. By affinity chromatography using rabbit anti hydatid fluid IgG coupled covalently to CNBr-Sepharose 4B a protein fraction (Em 1) containing shared antigens of both parasites could be isolated from an extract ofE. multilocularis metacestode tissue. From the same source another antigen fraction (Em 2) with a high degree of specificity forE. multilocularis was prepared by immunosorption. Antigen Em 1 was equally sensitive for the detection of antibodies againstE. granulosus andE. multilocularis, whereas antigen fraction Em 2 appeared to be more specific forE. multilocularis. A correct serological differential diagnosis was achieved in 95% of 57 confirmed cases of human cystic or alveolar echinococcosis by the simultaneous use of both antigen fractions in the ELISA and by comparison of their reactivities.     

Prévalence du téniasis échinococcique chez les chiens errants dans la région de Constantine,Nord-Est algérien

In North Africa, the domestic dog is regarded as the main reservoir for infection by Echinococcus granulosus of domestic livestock and man. In Algeria, there is very little data on the rate of infestation of dogs, while the prevalence of E. granulosus in the definitive host is a very reliable marker of the potential risk of transmission of cystic tapeworm to humans and livestock. To find out this information, a survey was conducted to assess the prevalence of infection with E. granulosus in stray dogs in the region of Constantine (North-East Algeria). We autopsied and examined 120 stray dogs, 22 (18.3%) of which were infected with E. granulosus, with an average intensity of infestation of 249 worms. The prevalence in the area of survey was evaluated: 15.5% (14/90) and 26.6% (8/30) dogs were parasitized by E. granulosus in urban and rural areas respectively. The influence of age on the rate of infection was very marked. In addition, the appreciation of the prevalence of parasitism by cestodes as a whole showed that 56 (46.6%) animals out of 120 were infected. Facing such a situation of endemic tapeworm parasitism, with a potential risk of transmission to humans, there is an urgent need to take measures to control and break the epidemiological cycles of the parasite.     

A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species

A hybridization probe-based real-time multiplex-nested PCR system was developed for the simultaneous detection of Echinococcus multilocularis and host species directly from faecal samples. Species identification was determined by melting curve analysis. Specificity was assessed by using DNA extracted from various cestodes (E. multilocularis, Echinococcus granulosus (G1), Echinococcus ortleppi, Echinococcus canadensis (G6, G7), Taenia crassiceps, Taenia hydatigena, Taenia mustelae, Taenia pisiformis, Taenia serialis, Taenia taeniaeformis, Mesocestoides leptothylacus), carnivores (Vulpes vulpes, Vulpes corsac, Vulpes ferrilata, Canis familiaris, Felis catus, Martes foina), Microtus arvalis and Arvicola terrestris. The analytical sensitivity was 10 fg, evaluated with serially diluted DNA of E. multilocularis to 10 μl total DNA solution from E. multilocularis-negative canid faeces. Based on a comparison of 47 dog samples from China, the proportion of the E. multilocularis-positive-tested samples by the real-time multiplex-nested PCR was moderately higher (38% vs. 30%) as when tested with a previously evaluated nested PCR with a sensitivity of 70–100%, depending on the number and gravidity status of worms present in the intestine (Dinkel et al., J Clin Microbiol 36:1871–1876, 1998). To assess the epidemiological applicability of this method, 227 canid faecal samples collected in the field were analysed. This newly developed real-time multiplex-nested PCR system is a specific, sensitive and reliable method for the detection of E. multilocularis and host species in faecal samples for epidemiological purposes.     

Effects of isoprinosine onEchinococcus multilocularis andE. granulosus metacestodes

An in vivo study of the effect of Isoprinosine onEchinococcus multilocularis andE. granulosus metacestodes (Cestoda) was performed. Short- and longterm treatments with different doses were tested in experimental hosts: jirds forE. multilocularis and mice forE. granulosus. Modifications in the weight of animals as well as macroscopical and ultrastructural aspects were registered for each animal group. The results obtained showed a considerable ultrastructural alteration inE. multilocularis metacestodes after short-term treatment with the highest dose, and the tissue transplants were negative. InE. granulosus cysts the ultrastructural damage was also very important and its severity increased as the dose was raised. For the two parasites, the macroscopical aspect and weight of the lesions were profoundly changed.     

Paramyosin ofEchinococcus granulosus: cDNA sequence and characterization of a tegumental antigen

A ZAPII cDNA library ofEchinococcus granulosus larvae was expressed inEscherichia coli SURE cells. Screening of the library with a rabbit antiserum raised against total larval antigen yielded several immunoreactive clones. For analysis of the nucleotide sequence, in vivo excision into pBlueskript was carried out and the 3 end of the cloned insert was sequenced. Three of these clones exhibited identical nucleotide sequences, suggesting expression of identical genes. The complete nucleotide sequence of the largest clone, EG36, with a 3.4-kb insert was determined, presenting an open reading frame of 2.59 kb. The predicted amino acid sequence showed 71.4% identity to theSchistosoma mansoni paramyosin and a significant homology to a 17 amino-acid peptide sequence from antigen B ofTaenia solium. From these data we conclude that EG36 is the paramyosin ofE. granulosus. For protein purification, the coding sequence of the cDNA was amplified by polymerase chain reaction and ligated in frame into the expression vector pGEX-3X. Affinity-chromatography-purified GST fusion protein was used to induce a polyclonal rabbit antiserum. Immunoblot analysis revealed the expression of a 97-kDa protein by theE. coli clone and that of a protein with a similar molecular weight in protoscolices fromE. granulosus andE. multilocularis as well as inE. granulosus cyst fluid. Immunofluorescence studies showed that EG36 was localized throughout the tegument ofE. granulosus andE. multilocularis larvae. Sera from patients suffering from echinococcosis, schistosomiasis, and neurocysticercosis reacted with the purified fusion protein when tested in an enzyme-linked immunosorbent assay.     

Effect of praziquantel on adultEchinococcus granulosus in vitro: Scanning electron microscopy

The effect of praziquantel in vitro at concentrations of 5, 50 and 500 ppm for 1 h resulted in the progressive breakdown of the tegument and in morphologic distortion of adultEchinococcus granulosus when compared to controls. Scanning electron microscopy of all specimens treated in the various concentrations of praziquantel showed loss of most, if not all, of the rostellar hooks and changes in the structure of the suckers. Many of the tapeworms immediately detached from the host's gut upon being placed in the drug, and all treated cestodes exhibited contraction or swelling, particularly in the penultimate proglottid. Intense contraction was apparent in the worms exposed to the higher drug concentrations. Characteristic conical microtriches on the terminal proglottid, as observed in the control specimens, became fused and matted when exposed to 5 ppm of praziquantel. At a drug concentration of 50 ppm, the tegumental surface developed grooves or furrows between clumps of fused microtriches, while 500 ppm caused production of holes within the denuded tegument of the parasite. Ovoid bodies, presumed to be eggs, were observed on the outer surfaces and just below the tegument of tapeworms treated with concentrations of 50 ppm. These structures also appeared to adhere to the outer surfaces of specimens exposed to 500 ppm. In view of the foregoing, special care should be taken in handling and disposing of feces from infected or suspect dogs after praziquantel treatment, since the breakdown in the tegumental surface ofE. granulosus presumably results in the release of potentially infective eggs.     

Echinococcus granulosus of camel origin: development in dogs and parasite morphology

The developmental characteristics ofEchinococcus granulosus of camel origin were studied in four dogs artificially infected with protoscolices originating from hydatid cysts isolated from the lung of a camel (Camelus dromedarius). Two dogs each were necropsied 34/35 and 41 days post-infection (p.i.); one dog had a low worm burden and the others were heavily infected (27,625–41,150 worms). At day 35 p.i., 20% of the parasites had developed three segments and the uterus of the vast majority of the total population was full of developing eggs in the terminal segment. At day 41, up to 58% of the parasites contained mature eggs (embryonated eggs with fully developed, thick-shelled embryophores). Morphological studies revealed the following major characteristics for 35 day-old worms: the mean length of the terminal segment accounted for 54% of the total worm length; the position of the sexually mature segment was always terminal; the female reproductive system possessed an elongated ovary with compact lobules; the female ducts were also compact; the Mehlis' gland was covered by the vitelline gland and the testes were distributed throughout the segment, with 1 row posterior to the vitelline gland. The camel isolate can readily be distinguished from the horse and sheep strains, but it is similar to the cattle strain in some respects, particularly in its precocious development. However, the camel isolate differs from the cattle strain in the position of the sexually mature segment, arrangement of the testes and structure of the female reproductive system. As in the cattle strain, the metacestodes in the principal intermediate host are mostly localised in the lung and have a high fertility rate. Detailed morphological characteristics and biological features of the sheep, cattle, horse and camel isolates ofE. granulosus are presented.Increasing evidence is being accumulated that local populations ofEchinococcus granulosus may vary in morphological and biological features and that the occurrence of intraspecific variants or strains can play an important role in the epidemiology of echinococcosis (Rausch 1986; McManus and Smyth 1986; Thompson 1986; Thompson 1988; Thompson and Allsopp 1988; Eckert and Thompson 1988), because strains differ in their infectivity to final and intermediate hosts and their transmission patterns. Moreover, the pathogenic significance of strains to humans may also vary.The camel is an important host ofE. granulosus and is commonly infected throughout Africa and the Middle East (Schwabe 1986). The camel has attracted much interest as an intermediate host ofE. granulosus, particularly in its role as a reservoir of infection in humans. In addition, it has been proposed that the camel form ofE. granulosus may be a different subspecies or strain (Pandey et al. 1986). However, very little comparative work has been carried out to characterise isolates ofE. granulosus from camels (Thompson and Lymbery 1988).The aim of the present work was to study the morphological and biological characteristics of intestinal stages ofE. granulosus of camel origin obtained from artificially infected dogs. In addition, data were collected on the metacestodal stage.     

Echinococcus granulosus infection of farm dogs of Iran

The prevalence and distribution of Echinococcus granulosus in sheepdogs was studied in 13 provinces of Iran, where 90% of the Iranian sheep and goat populations and, thus, sheepdogs are found. Worms were found in 27.17% of 390 dogs successfully purged with 4 mg/kg arecoline hydrobromide. The highest prevalence was detected in dogs from the rural areas of Isfahan (central part of Iran) and the lowest, in dogs from those of Sistan (Southeast Iran). The frequency distribution of E. granulosus was overdispersed, with only a few animals harboring heavy infections. Received: 2 July 1997 / Accepted: 10 September 1997     

Production and immunoanalytical application of 32 monoclonal antibodies against metacestode somatic antigens of Echinococcus multilocularis

Alveolar echinococcosis is a rare but potentially fatal disease. Immunodiagnosis based on antibodies or antigens plays an important role in its diagnosis. In this study, metacestode somatic antigens of Echinococcus multilocularis were used to immunize BALB/c mice, and hybridomas were formed by cell fusion. Making use of the inherent effect of monoclonal antibody techniques to isolate different epitopes, we obtained a repertoire of 32 monoclonal antibodies against the metacestode somatic antigens. These monoclonal antibodies were used to investigate the specificity and localization of the metacestode antigens by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Nine antibodies specifically reacted with E. multilocularis, while 14 and ten cross-reacted with Echinococcus granulosus and Taenia saginata, respectively. Twenty-five antibodies stained the laminated layer. Eight reacted with the tegument of the protoscolex. Fourteen antibodies recognized the germinal layer. Most of the monoclonal antibodies can react with the antigen Em2. One antibody can react with antigen Em2 and Em10. One antibody that cross-reacted with T. saginata stained the germinal layer and protoscolex, especially its hooklets and suckers, but could not react with Em2 and Em10 antigens. It detected protein bands at 26 and 52 kDa. Two E. multilocularis-specific monoclonal antibodies stained both the germinal and laminated layers and could be used not only to purify specific antigens but also for immunohistochemical studies of E. multilocularis. In summary, these 32 monoclonal antibodies could have potential applications as useful tools in further studies of E. multilocularis antigen profiles.     

Analysis of taeniid antigens using monoclonal antibodies toEchinococcus granulosus antigen 5 and antigen B

Antigens derived fromEchinococcus granulosus, Taenia hydatigena andT. pisiformis cyst fluids,T. solium cysticerci,E. multilocularis protoscoleces andE. vogeli cyst membranes were examined by enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis (IEP) using four monoclonal antibodies (mAb) toE. granulosus antigen 5 (Ag5) and antigen B (AgB). Anti-Ag5 mAbs 24.14 and 61A12 reacted strongly withT. hydatigena andT. pisiformis cyst fluids and, to a lesser degree, anti-AgB MAbs 31.15 and 39B3 also displayed some reaction with these antigens in ELISA. The formation of a modified arc 5 band between Anti-Ag5 mAbs andT. hydatigena cyst fluid (THCF) in IEP further confirms the existence of Ag5 inT. hydatigena cyst fluid. However, the inability of THCF andT. pisiformis cyst fluid (TPCF) to form an AgB band as well as that of TPCF to form an arc 5 band with mAbs in IEP does not exclusively prove the lack of AgB in THCF and TPCF or the lack of Ag5 in TPCF. The absence of a reaction of mAbs withT. solium, E. multilocularis andE. vogeli antigen preparations in ELISA or IEP would suggest that these mAbs may recognise epitopes different from those ofT. solium, E. multilocularis andE. vogeli parasites; this might be exploited for specific differentiation ofE. granulosus.     

The EmsB Tandemly Repeated Multilocus Microsatellite: a New Tool To Investigate Genetic Diversity of Echinococcus granulosus Sensu Lato

Cystic echinococcosis (CE) is a widespread and severe zoonotic disease caused by infection with the larval stage of the eucestode Echinococcus granulosus sensu lato. The polymorphism exhibited by nuclear and mitochondrial markers conventionally used for the genotyping of different parasite species and strains does not reach the level necessary for the identification of genetic variants linked to restricted geographical areas. EmsB is a tandemly repeated multilocus microsatellite that proved its usefulness for the study of genetic polymorphisms within the species E. multilocularis, the causative agent of alveolar echinococcosis. In the present study, EmsB was used to characterize E. granulosus sensu lato samples collected from different host species (sheep, cattle, dromedaries, dogs, and human patients) originating from six different countries (Algeria, Mauritania, Romania, Serbia, Brazil, and the People''s Republic of China). The conventional mitochondrial cox1 and nad1 markers identified genotypes G1, G3, G5, G6, and G7, which are clustered into three groups corresponding to the species E. granulosus sensu stricto, E. ortleppi, and E. canadensis. With the same samples, EmsB provided a higher degree of genetic discrimination and identified variations that correlated with the relatively small-scale geographic origins of the samples. In addition, one of the Brazilian single hydatid cysts presented a hybrid genotypic profile that suggested genetic exchanges between E. granulosus sensu stricto and E. ortleppi. In summary, the EmsB microsatellite exhibits an interesting potential for the elaboration of a detailed map of the distribution of genetic variants and therefore for the determination and tracking of the source of CE.Cystic echinococcosis (CE) is a widespread and severe zoonosis caused by infection with the larval stage of the eucestode Echinococcus granulosus sensu lato. Classification of the organisms within this paraphyletic taxon has undergone and continues to undergo important changes. Mitochondrial DNA-based studies have shown that E. granulosus sensu lato is composed of 10 heterogeneous groups of variants, defined as strains (strains G1 to G10) (5, 6, 17). However, these strains are now reorganized within distinct species (21, 25). E. granulosus sensu stricto encompasses strains G1, G2, and G3; E. equinus corresponds to strain G4; and E. ortleppi comprises strain G5. Strains G6, G7, G8, G9, and G10 have been also classified under a well-supported monophyletic species, E. canadensis (16, 19, 21). Recently, the lion strain has been characterized as another new species, E. felidis (11).Currently, mitochondrial and nuclear markers are not sufficiently polymorphic for use for the identification of genetic variations that could reflect geographically based peculiarities. The use of sensitive tools such as microsatellites may provide more information about the polymorphism of the parasite and the spatial-temporal characteristics of its patterns of transmission between foci. However, to date, only four single-locus microsatellites have been used to investigate E. granulosus sensu lato isolates: U1snRNA, EgmSca 1, EgmSca 2, and EgmSga 1. The U1snRNA gene exhibited 11 distinct profiles: 8 for E. granulosus sensu stricto (G1/G2), 2 for E. ortleppi (G5), and 1 for E. canadensis (G6) (22). However, no spatial correlation with the geographic origin of the isolates was observed. Among the three microsatellites described by Bartholomei-Santos et al. (4), only EgmSca 1 correlated the genotypes with the origins of the samples.Recently, Bart et al. (3) developed EmsB, a tandemly repeated multilocus microsatellite, for the genotyping of E. multilocularis. This marker showed a higher level of intraspecific variability compared with that shown by any previously published marker, as well as a very high degree of sensitivity (7, 13-15). It is composed of an array of 800-bp DNA fragments containing a variable combination of CA and GA repeats. The use of such a microsatellite could contribute to the better identification of the spatial-temporal characteristics of the E. granulosus transmission patterns, as is the case for E. multilocularis. Indeed, using this new tool, Knapp et al. managed to perform efficient genetic tracking of E. multilocularis isolates in different foci of alveolar echinococcosis (13-15). Furthermore, because of its localization within the nuclear genome, cross-fertilization processes may modify EmsB patterns. Therefore, this microsatellite may be an interesting marker for use for both assessment of the genetic polymorphism of E. granulosus sensu lato and detection of the genetic exchange events between the variants.In the present work, we tackled its variability using a panel of 127 E. granulosus sensu lato samples collected in six countries where CE is endemic.