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.     

Cloning and immunological characterisation ofEchinococcus granulosus ferritin

A cDNA clone ofEchinococcus granulosus has been isolated from an expression library screened with sera from cystic echinococcosis patients. The deduced amino acid sequence shows 56% homology to the heavy chain of human ferritin.E. granulosus ferritin contains 173 amino acid residues and has a calculated molecular weight of 19830 Da and a statistical isoelectric point of 7.6. Functionally important amino acid residues of the ferroxidase centre are conserved in comparison with other ferritins. In vitro-translatedE. granulosus ferritin was tested for its diagnostic potential by immunoprecipitation. The antigenic reactivity exhibited a good potential for the further development ofE. granulosus ferritin as an immunodiagnostic tool for human hydatidosis.     

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.     

Prevalence survey and first molecular characterization of Echinococcus granulosus in France

Human cystic echinococcosis (hydatid disease) caused by the Echinococcus granulosus tapeworm continues to be a substantial cause of morbidity and mortality in many parts of the world. France is still considered as endemic area, but the current infestation by E. granulosus of intermediate hosts in France remains currently unknown due to the absence of official data reporting for the last 20 years. A 1-year prevalence survey was conducted in the 24 slaughterhouses of ten departments of the South of France. We demonstrate that the E. granulosus parasite is still currently present at low prevalence at slaughterhouses in the study area (4 cases for 100,000 sheep and 3 cases for 100,000 cattle). In addition, we assess the presence of genotype G1 in infected animals and identify for the first time in France genotypes G2 and G3 of E. granulosus sensu stricto.     

Molecular characterization of Echinococcus granulosus in south-eastern Romania: evidence of G1–G3 and G6–G10 complexes in humans

Echinococcus granulosus is the aetiological agent of cystic echinococcosis (CE), which is a public health problem in many eastern European countries, particularly in Romania, where the infection causes a high number of human and animal cases. To shed light on the transmission patterns of the parasite, we performed a genotyping analysis on 60 cyst samples obtained from patients who live in south-eastern Romania and who underwent surgery for liver or lung CE. DNA was extracted from the endocysts or the cyst fluids, and fragments of cytochrome c oxidase subunit 1 and NADH dehydrogenase subunit 1 mitochondrial genes (cox1 and nd1, respectively) were amplified by PCR and sequenced. We found that most of the samples analysed (59/60) belonged to the G1–G3 complex (E. granulosus sensu stricto), which contains the most widespread and infective strains of the parasite. We also identified the first human patient infected by a non-G1–G3 genotype of E. granulosus in this country. As the DNA sequence of this cyst sample showed maximum homology with the G6–G10 complex (Echinococcus canadensis), this is, in all likelihood, a G7 genotype, which is often found in pigs and dogs in most countries of eastern and south-eastern Europe.     

Prevalence and diversity of cystic echinococcosis in livestock in Maasailand, Kenya

Cystic echinococcosis (CE) is a zoonotic disease caused by several members of the Echinococcus granulosus species complex. In East Africa, several species/strains are known to occur in livestock and humans, but host preferences, relative frequencies and spatial distribution of these taxa are poorly known. Here, we contribute livestock data for Maasailand of southern Kenya. Total CE prevalence was 25.8?% in cattle (151/587), 16.5?% in sheep (71/430) and 10.8?% in goats (21/194), which is a significant increase compared to surveys done about three decades ago. The majority of cysts occurred in the liver (56?% in cattle, 70?% in sheep and 65?% in goats). Molecular characterization by PCR?CRFLP and sequencing of parts of the mitochondrial nad-1 gene was done for a subsample of 285 cysts. E. granulosus G1 was dominant in all host species (200 of 201 cysts from cattle, 68 of 69 from sheep and 11 of 15 from goats); the remaining taxa were Echinococcus canadensis G6 (one cyst from sheep, four from goats) and Echinococcus ortleppi (one cyst from cattle). Considering cyst fertility, sheep appear to be the most important hosts for E. granulosus G1, while goats were found to be suitable hosts for E. canadensis G6 (three of four cysts were fertile). For the first time, E. ortleppi was found in cattle from southern Kenya. Our data show an intense and possibly increasing level of CE transmission in southern Kenya, and the predominance of E. granulosus G1, which appears to be particularly pathogenic to humans, calls for urgent control measures.     

Diagnosis of primary hydatid cyst of thigh by fine needle aspiration cytology

Hydatidosis is a parasitic infestation caused by larval form of the tapeworm, Echinococcus granulosus. Primary hydatid cyst in the skeletal muscles and subcutaneous tissue of thigh without involving thoracic and abdominal organs is an exceptional entity, even in countries where the Echinococcus infestation is endemic. We report an unusual case of primary hydatid cyst of thigh in proximity to skeletal muscles. This case illustrates that echinococcal disease should be considered in the differential diagnosis of every subcutaneous cystic mass. This case is presented here for its rarity.     

Application of recombinant Echinococcus granulosus antigen B to ELISA kits for diagnosing hydatidosis

Echinococcus granulosus causes human cystic echinococcosis as an important public health problem in many regions of the world. There are some problems in primary diagnosis such as cross-reaction with sera from patients with other parasitic disease in serological tests. The use of an appropriate source of antigenic material is a very important and crucial point in the improvement of the serodiagnostic features such as enzyme-linked immunosorbent assay (ELISA) method. We expressed and purified recombinant AgB of Echinococcus granulosus and used as antigen in ELISA method. Serum samples were given from 36 cystic hydatid disease patients that have been confirmed by surgical operation as well as 36 healthy individuals sera were tested by ELISA method using recombinant AgB and compared with commercial kit (Euroimmun) for specificity and sensitivities value. The sensitivity of 91.66% and specificity of 97.22% were determined by homemade kit.     

High resolution melting technique for molecular epidemiological studies of cystic echinococcosis: differentiating G1, G3, and G6 genotypes of Echinococcus granulosus sensu lato

Reliable and rapid genotyping of large number of Echinococcus granulosus sensu lato isolates is crucial for understanding the epidemiology and transmission of cystic echinococcosis. We have developed a method for distinguishing and discriminating common genotypes of E. granulosus s.l. (G1, G3, and G6) in Iran. This method is based on polymerase chain reaction coupled with high resolution melting curve (HRM), ramping from 70 to 86 °C with fluorescence data acquisition set at 0.1 °C increments and continuous fluorescence monitoring. Consistency of this technique was assessed by inter- and intra-assays. Assessment of intra- and inter-assay variability showed low and acceptable coefficient of variations ranging from 0.09 to 0.17 %. Two hundred and eighty E. granulosus s.l. isolates from sheep, cattle, and camel were used to evaluate the applicability and accuracy of the method. The isolates were categorized as G1 (93, 94, and 25 %), G3 (7, 4, and 4 %), and G6 (0, 2, and 71 %) for sheep, cattle, and camel, respectively. HRM results were completely compatible with those obtained from sequencing and rostellar hook measurement. This method proved to be a valuable screening tool for large-scale molecular epidemiological studies.     

Cystic echinococcosis in Turkey: genetic variability and first record of the pig strain (G7) in the country

A sample of 22 Echinococcus granulosus isolates collected from 12 sheep and ten humans from a focus of cystic echinococcosis in western Turkey was examined by DNA sequencing of four mitochondrial genes (cox1, atp6, nad1, rrnS). Results demonstrated the presence of two species of E. granulosus complex, E. granulosus sensu stricto and E. canadensis. Of E. granulosus sensu stricto, the G1 genotype (including three microvariants) was found in 17 isolates from humans and sheep, the G3 genotype and an intermediate form G1/G3 in one isolate each (both from sheep). Of E. canadensis, the pig strain G7 was found in three isolates from sheep and human. This is the first report of this strain in Turkey. Its presence has implications for local control programs due to its shorter maturation rate in dogs compared with E. granulosus sensu stricto. Goat and/or wild boar are likely reservoirs for G7 in the region. We provided further data on the pattern and frequency of nucleotide substitutions within the G1/G3 cluster. Based on our results and GenBank records, G2 (Tasmanian sheep strain) is not considered as a discrete genotypic unit, as its sequences at polymorphic sites conform to microvariants of both G1 and (more often) G3.     

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.

     

Epidemiological studies on Echinococcus in Pampas fox (Lycalopex gymnocercus) and European hare (Lepus europaeus) in Buenos Aires province, Argentina

In Argentina, hydatid disease caused by Echinococcus granulosus is widespread. The south of Buenos Aires province, Argentina, is one of the three regions where hydatidosis is endemic. Although domestic dogs and sheep are considered to be the main hosts for E. granulosus, the potential role of wildlife in the local transmission of E. granulosus has not been investigated. The aim of this study was to estimate the hydatidosis/echinococcosis prevalence in European hare (Lepus europaeus) and Pampas fox (Lycalopex gymnocercus), two abundant species with a strong predator–prey relationship in rural areas of Buenos Aires province using different diagnostic tests. A total of 61 fox intestines were examined, finding that 52 (85.2 %) harbored at least one helminth species. However, no adult or immature form of Echinococcus sp. was found in the intestinal contents. Coproparasitological analysis and Copro–ELISA followed by Copro–PCR were used as supplementary diagnostic tests. Only one (1.7 %) of 59 fecal samples was positive to Taeniidae eggs by coproparasitological analysis, but this same sample was negative by the Copro–ELISA test. The analysis by Copro–ELISA showed 6 of 57 (10.6 %) positive samples, but the Copro–PCR tests carried out on these samples were negative to E. granulosus. A total of 6,808 lungs, 3,576 livers, and 3,542 hearts of hunted hares were examined and palpated, but no structure resembling hydatid cysts were detected. Our results suggest that hares and Pampas foxes are not currently important wild reservoirs of E. granulosus in the studied area.     

In vitro and in vivo treatments of Echinococcus granulosus with Huaier aqueous extract and albendazole liposome

The aim of this study was to investigate the in vitro and in vivo efficacies of chemotherapy employing albendazole liposome (L-ABZ), Huaier aqueous extract, and a Huaier aqueous extract/L-ABZ combination against Echinococcus granulosus. Protoscolices of E. granulosus were incubated in vitro with the two drugs, either separately or in combination, at the following final concentrations: 2 mg/mL Huaier aqueous extract, 10 μg/mL L-ABZ, and 2 mg/mL Huaier aqueous extract + 10 μg/mL?L-ABZ. Huaier aqueous extract and L-ABZ displayed slower protoscolicidal activity when applied separately than when used in combination. The maximum protoscolicidal effect was found with the combination Huaier aqueous extract + L-ABZ. Despite the low Huaier aqueous extract + L-ABZ concentrations used, protoscolex viability dropped rapidly. In vivo studies were performed on mice injected with protoscolices of E. granulosus. Huaier aqueous extract and L-ABZ were administered three times a week for a period of 4 months by the oral route. Huaier aqueous extract in E. granulosus-infected mice was effective. Combined application of both drugs did increase the treatment efficacy. In conclusion, the outcomes obtained clearly demonstrated that in vitro and in vivo treatment with Huaier aqueous extract and L-ABZ is effective against E. granulosus.     

First detection and molecular characterization of Echinococcus equinus in a Mule in Turkey

Cystic echinococcosis is a zoonotic disease with a cosmopolital distribution. It is caused by the larval stages (metacestodes) of the parasite Echinococcus granulosus which infects different animal species. In this report, we present a case of E. granulosus infection in a mule and molecular characterization of the cyst. For this purpose parasite material was collected from the liver of a necropsied mule. DNA was isolated and PCR amplification of mitochondrial 12S rRNA as well as partial sequencing of mitochondrial cytochrome c oxidase subunit 1 (mt-CO1) genes were performed. Six unilocular cysts, filled with clear fluid were found in the liver and spleen. All cysts were found to be fertile. The 12S rRNA-PCR did not yield any band while mt-CO1-PCR yielded a 446 bp sized amplification product. Sequence corresponding to mt-CO1 gene was identical to a sequence reported for E. equinus (formerly G4) (Genbank accession number: KC953029). This is the first record of E. equinus as a cause of cystic echinococcosis in a mule in Turkey.     

<Emphasis Type="Italic">Echinococcus granulosus</Emphasis> strain typing in Bulgaria: the G1 genotype is predominant in intermediate and definitive wild hosts

Addressing the genetic variability in Echinococcus granulosus is epidemiologically important, as strain characteristics may influence the local transmission patterns of zoonotic cystic echinococcosis. To classify the genotype(s) present in intermediate (pig, cattle and sheep) and definitive (jackal and wolf) hosts in Bulgaria, a DNA-based approach was used to assess parasite protoscoleces or strobiles. Genes corresponding to coding and non-coding regions of the nuclear and mitochondrial genome (ND-1, HBX, Act II, AgB-1) were amplified by PCR and subsequently sequenced. The sequences resolved were all found to be identical to those published for the common sheep strain of E. granulosus, indicating that the G1 genotype is predominant in Bulgaria. One microvariant for ND-1 was found in the pig isolates; however no epidemiological significance was attributed to this finding.     

Observations onEchinococcus multilocularis in the definitive host

Six dogs were found to be susceptible to experimental infections with a European isolate ofEchinococcus multilocularis from southern Germany. Two cats were only poorly susceptible. Adult worms were not evenly distributed throughout the small intestine and the majority of parasites were found in the posterior region. The mode of attachment ofE. multilocularis in the dog was similar to that forE. granulosus with the adult worm extending its rostellum deep within a crypt of Lieberkühn. In cats only few worms were found to have penetrated deeply between the villi.E. multilocularis was found to possess a modified group of rostellar tegumental cells, morphologically and functionally identical to those described inE. granulosus and previously referred to as the “rostellar gland”. By studying development in vivo and in vitro, the time required for the production of shelled eggs was demonstrated to be only 28 days. Concurrent experimental infections in dogs withE. multilocularis andE. granulosus revealed that both species will develop together in the same host. Their development was not retarded in any way by the presence of the other and both species were able to coexist in the same area of the intestine.     

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.     

Differential diagnosis of cystic and alveolar echinococcosis using an immunochromatographic test based on the detection of specific antibodies

Human cystic and alveolar echinococcoses are zoonotic diseases caused by the larval stages of Echinococcus granulosus and Echinococcus multilocularis, respectively. As the diseases are co-endemic in many areas of the world, a simple and rapid test for the differential diagnosis of cystic echinococcosis (CE) and alveolar echinocoocosis (AE) is needed. Here, we describe the development of an immunochromatographic test (ICT) using crude hydatid cyst fluid and a recombinant 18-kDa protein (rEm18) as antigens for the detection of E. granulosus and E. multilocularis antibodies in serum samples. The ICT was evaluated with serum samples from 195 echinococcosis patients from different endemic areas in northwestern China. These included 144 from CE patients, 51 from AE patients, 67 from patients with other parasitic diseases, 13 from patients with serous hepatic cysts, and 60 from healthy individuals. The sensitivity and specificity of the ICT for CE were 91.0 and 96.9 % and for AE were 98.0 and 99.3 % with diagnostic efficiencies of 94.1 and 99.1 %, respectively. No significant differences and high degrees of agreement were found between the ICT and an enzyme-linked immunosorbent assay for both CE and AE. Five serum samples from cysticercosis patients and one serum sample from a healthy control were found positive for CE with the ICT. These findings indicate that this test allows for discrimination between both forms of human echinococcosis. In conclusion, the ICT developed in this study is a promising tool for the simultaneous detection and discrimination of CE and AE. This test will be useful for serodiagnosis of CE and AE in clinical settings and screening programs.     

Echinococcus spp. in central Kenya: a different story

Research on cystic echinococcosis (CE) has a long history in Kenya, but has mainly concentrated on two discrete areas, Turkana and Maasailand, which are known to be foci of human CE in Africa. Here, we report on a survey for CE in livestock from central to northeastern Kenya, from where no previous data are available. A total of 7,831 livestock carcasses were surveyed. CE prevalence was 1.92 % in cattle (n?=?4,595), 6.94 % in camels (n?=?216), 0.37 % in goats (n?=?2,955) and 4.62 % in sheep (n?=?65). Identification of the parasite was done using an RFLP-PCR of the mitochondrial nad1 gene, which had been validated before against the various Echinococcus taxa currently recognized as distinct species. From a total of 284 recovered cysts, 258 could be identified as Echinococcus granulosus sensu stricto (n?=?160), E. ortleppi (n?=?51) and E. canadensis (n?=?47) by RFLP-PCR of nad1. In cattle, fertile cysts occurred mostly in the lungs and belonged to E. ortleppi (31 of 54), while the vast majority were sterile or calcified cysts of E. granulosus s.s.. Most fertile cysts in camels belonged to E. canadensis (33 of 37); sterile or calcified cysts were rare. Goats harboured fertile cysts of E. ortleppi (n?=?3)—which is the first record in that host species—and E. canadensis (n?=?1), while all cysts of E. granulosus were sterile. Only sterile cysts were found in the three examined sheep. Typically, all cysts in animals with multiple infections belonged to the same species, while mixed infections were rare. Our data indicate that the epidemiological situation in central to northeastern Kenya is clearly different from the well-studied pastoral regions of Turkana and Maasailand, and the apparently low number of human CE cases correlates with the infrequent occurrence of E. granulosus s.s.     

An immunoblot for detection of Taenia saginata cysticercosis

Control measures to prevent human infections with the food-borne zoonotic helminth Taenia saginata are currently based on meat inspection, which shows rather low diagnostic sensitivity. To develop an immunoblot for detection of T. saginata-infected cattle, crude proteins of T. saginata cysts were extracted and separated with SDS-PAGE. The cyst antigens showed ten protein bands ranging from 260 to 14 kDa. T. saginata cyst proteins 260, 150, 130, 67, 60, 55, 50, and 23 kDa were immunoreactive with known positive sera of T. saginata-infected cattle but cross-reacted with sera from Echinocccus granulosus-infected ruminants. By contrast, 14- and 18-kDa cyst proteins reacted specifically with T. saginata-positive sera and thus might be potential candidates for the development of a T. saginata-specific immunoassay. Proteins of E. granulosus cysts and Taenia hydatigena cysts were also extracted and separated with SDS-PAGE. E. granulosus cysts revealed 11 protein bands ranging from 260 to 23 kDa. E. granulosus protein 60 kDa was immunoreactive with E. granulosus-positive sera only. The cyst of T. hydatigena showed 11 protein bands ranging from 290 to 14 kDa. The protein band 35 kDa showed cross-reaction with positive sera from both T. saginata- and E. granulosus-infected animals. A protein of 67 kDa was present in all three tested cestode species and was the major antigenic protein detected by sera of T. saginata- and E. granulosus-infected animals. Therefore, this protein represents a potential vaccine candidate against both cysticercosis and cystic echinococcosis in cattle.