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.     

Further molecular discrimination of Spanish strains of Echinococcus granulosus

We have designed two polymerase chain reaction (PCR) primer sets (PEg9F1-PEg9R1 and PEg16F1-PEg16R1) and two PCR protocols (Eg9-PCR and Eg16-PCR) for discrimination of Echinococcus granulosus genotypes. The oligonucleotide sequences originate from two E. granulosus DNA multiplex-PCR amplification fragments, previously reported, that allows species-specific discrimination between Taenia saginata, Taenia solium, and E. granulosus. The Eg9-PCR, Eg16-PCR, and Eg9-PCR linked restriction fragment length polymorphism (RFLP) analysis was used to characterize 53 E. granulosus isolates from the central region of Spain, highly endemic for echinococcosis. The analysis resulted in: (i) the discrimination of E. granulosus from Echinococcus multilocularis; (ii) the characterisation and discrimination of discrete E. granulosus strains from Spain; and (iii) the identification of two distinct genotypes within E. granulosus Spanish pig isolates. To further characterize the genetic variants in pigs, fragments of the NADH dehydrogenase I (ND1) and the cytochrome c oxidase subunit I (CO1) genes were amplified from parasite DNA and sequenced. The results again revealed the presence of two distinct genotypes: the G1 (sheep-dog strain) and G7 (pig-dog strain) genotypes. This observation could have important consequences for human health in Spain. Furthermore, the Eg9-PCR, Eg16-PCR, and Eg9-PCR-RFLP protocols can be used as additional methods to discriminate various E. granulosus genotypes.     

Molecular evidence for the presence of a G7 genotype of Echinococcus granulosus in Slovakia

Variability in Echinococcus granulosus is very important epidemiologically since strain characteristics may influence local patterns of transmission of hydatid disease. To classify the genotype presented in pig protoscoleces of the Slovak territory, a DNA-based approach has been used. Nucleotide sequences for a 471 bp region of the mitochondrial NADH dehydrogenase 1 (ND1) gene revealed a substantial affinity of isolates examined to the G7 genotype. Only a 0.9-3.4% sequence variation was recorded for E. granulosus samples compared with the reference G7 variant. To distinguish between G7 and G9 genotypes not differing in ND1 sequences, isolates were additionally examined by PCR-RFLP analysis of the nuclear ITS1 region. The resulting two-banded pattern is characteristic for the G7 strain. The data presented thus provides the first explicit evidence of the G7 genotype in the Slovak region.     

Growth and genotypes of Echinococcus granulosus found in cattle imported from Australia and fattened in Japan

At the abattoir on study in Miyazaki, Japan, 9537 imported cattle from Australia in average were slaughtered annually in the last 5 years (2006 to 2010) and hydatid cysts were constantly detected in about 1.8% of the cattle. In order to assess the risk of Echinococcus granulosus delivered to Japan by imported cattle, 250 cysts found in 103 cattle at the abattoir were examined for their biological characteristics and genotypes. The cattle slaughtered were imported from Australia at an age of 10-12 months old and fattened for 17-18 months in Japan. The cysts showed their size ranging from 4 to 108 mm and were mainly found in the lung. Mature protoscoleces were detected in the three largest cysts, all were of the G1 genotype. Most of the other cysts contained clear cyst fluid and had thin laminated layer with no protoscoleces. The finding implies a potential risk of E. granulosus being established in Japan, thus strict and proper meat inspection and consequent offal condemnation are requisite at abattoirs that deal with imported cattle. Genotyping based on partial fragments of mitochondrial cox1, rrnS and nad1 genes were performed on the 66 cysts, showing that most of the cysts were G1 genotype (common sheep strain). However, two and four cysts were considered as G2 (Tasmanian sheep strain) and G3 (buffalo strain) genotypes, respectively. Since it has been widely recognized that G1 is the only genotype distributing in mainland Australia and that G2 genotype has been eradicated from Tasmania, the finding of those genotypes from Australian cattle indicated that certain genotypes other than G1 genotype are distributing in mainland Australia.     

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.     

Echinococcus granulosus: DNA extraction from germinal layers allows strain determination in fertile and nonfertile hydatid cysts

A method for the isolation of Echinococcus granulosus DNA from germinal layers of hydatid cysts is described. The method includes a hexadecyltrimethylammonium bromide/chloroform extraction and an adsorption to diatomaceous earth suspension. DNA suitable for polymerase chain reaction was obtained and used for parasite strain determination by mitochondrial cytochrome c oxidase I gene sequencing. Fertile and nonfertile cyst isolates from sheep, cattle, pigs, and humans were characterized. Hitherto, no direct parasite strain characterization has been made on nonfertile hydatid cysts, whereas here we report that nonfertile hydatid cysts were produced by sheep strain (G1 genotype) in sheep, cattle, and humans and by pig strain (G7 genotype) in pigs.     

Genetic Characterization of Human-Derived Hydatid Cysts of Echinococcus granulosus Sensu Lato in Heilongjiang Province and the First Report of G7 Genotype of E. canadensis in Humans in China

Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus sensu lato (s.l.) is one of the most important zoonotic parasitic diseases worldwide and 10 genotypes (G1–G10) have been reported. In China, almost all the epidemiological and genotyping studies of E. granulosus s.l. are from the west and northwest pasturing areas. However, in Heilongjiang Province of northeastern China, no molecular information is available on E. granulosus s.l. To understand and to speculate on possible transmission patterns of E. granulosus s.l., we molecularly identified and genotyped 10 hydatid cysts from hepatic CE patients in Heilongjiang Province based on mitochondrial cytochrome c oxidase subunit I (cox1), cytochrome b (cytb) and NADH dehydrogenase subunit 1 (nad1) genes. Two genotypes were identified, G1 genotype (n = 6) and G7 genotype (n = 4). All the six G1 genotype isolates were identical to each other at the cox1 locus; three and two different sequences were obtained at the cytb and nad1 loci, respectively, with two cytb gene sequences not being described previously. G7 genotype isolates were identical to each other at the cox1, cytb and nad1 loci; however, the cytb gene sequence was not described previously. This is the first report of G7 genotype in humans in China. Three new cytb gene sequences from G1 and G7 genotypes might reflect endemic genetic characterizations. Pigs might be the main intermediate hosts of G7 genotype in our investigated area by homology analysis. The results will aid in making more effective control strategies for the prevention of transmission of E. granulosus s.l.     

Occurrence of the G3 Indian buffalo strain of Echinococcus granulosus in cattle

During a survey carried out to define the occurrence of Echinococcus granulosus in cattle bred in the province of Rieti (Central Italy), molecular diagnostics (PCR amplification and sequencing of a partial region of the mitochondrial CO1 gene) showed that 6/10 positive bovines harboured hydatid cysts (No.=16) genetically identical (95.8-100%) to the Indian buffalo genotype G3. As far the location of the 16 cysts, 11 of them were found in the lungs of three animals, whereas 5 cysts were in the liver of three parasitized hosts. The occurrence of genotype G3 in 60% of parasitized bovines living in an area never studied before provides more definite evidence about the existence of the strain in this region, and proves that cattle have to be considered a non-accidental host.     

Immunity and vaccine control of Echinococcus granulosus infection in animal intermediate hosts

Much progress has been made with characterisation of the EG95 vaccine which can be used to prevent hydatid infection in animal intermediate hosts of Echinococcus granulosus. The vaccine comprises a single recombinant oncosphere antigen and the adjuvant Quil A. It induces complement-fixing antibodies that kill the invading oncosphere early in an infection. In the majority of vaccinated animals, no hydatid cysts occur following a challenge infection. However, a small number of viable cysts may occur in some vaccinated animals. The vaccine has proved effective in vaccine trials carried out in sheep in New Zealand, Australia, Argentina, Chile and China as well as in goats and cattle. Investigations of the genetic diversity of the gene encoding EG95 have identified no unequivocal variation within the G1 strain parasites; however DNA sequence diversity within the EG95 family of genes has been found in G6/G7 parasites. GMP production scale-up of the vaccine has been undertaken in New Zealand and China and it is expected that the vaccine will be become available through these sources for implementation as part of hydatid control programs worldwide.     

Molecular Characterization of Echinococcus granulosus Sensu Lato from Farm Animals in Egypt

Little is known on the diversity and public health significance of Echinococcus species in livestock in Egypt. In this study, 37 individual hydatid cysts were collected from dromedary camels (n=28), sheep (n=7) and buffalos (n=2). DNA was extracted from protoscoleces/germinal layer of individual cysts and amplified by PCR targeting nuclear (actin II) and mitochondrial (COX1 and NAD1) genes. Direct sequencing of amplicons indicated the presence of Echinococcus canadenesis (G6 genotype) in 26 of 28 camel cysts, 3 of 7 sheep cysts and the 2 buffalo derived cysts. In contrast, Echinococcus granulosus sensu stricto (G1 genotype) was detected in one cyst from a camel and 4 of 7 cysts from sheep, whereas Echinococcus ortleppi (G5 genotype) was detected in one cyst from a camel. This is the first identification of E. ortleppi in Egypt.     

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.     

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).     

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.     

Molecular characterization of Echinococcus granulosus from Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene

Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosus worldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for 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.     

Echinococcus granulosus: variability of the host-protective EG95 vaccine antigen in G6 and G7 genotypic variants

Cystic hydatid disease in humans is caused by the zoonotic parasite Echinococcus granulosus. As an aid to control transmission of the parasite, a vaccine has been produced for prevention of infection in the parasite’s natural animal intermediate hosts. The vaccine utilizes the recombinant oncosphere protein, EG95. An investigation into the genetic variability of EG95 was undertaken in this study to assess potential antigenic variability in E. granulosus with respect to this host-protective protein. Gene-specific PCR conditions were first established to preferentially amplify the EG95 vaccine-encoding gene (designated eg95-1) from the E. granulosus genome that also contains several other EG95-related genes. The optimized PCR conditions were used to amplify eg95-1 from several parasite isolates in order to determine the protein-coding sequence of the gene. An identical eg95-1 gene was amplified from parasites showing a G1 or G2 genotype of E. granulosus. However, from isolates having a G6 or G7 genotype, a gene was amplified which had substantial nucleotide substitutions (encoding amino acid substitutions) compared with the eg95 gene family members. The amino acid substitutions of EG95 in the G6/G7 genotypes may affect the antigenicity/efficacy of the EG95 recombinant antigen against parasites of these genotypes. These findings indicate that characterization of eg95 gene family members in other strains/isolates of E. granulosus may provide valuable information about the potential for the EG95 hydatid vaccine to be effective against E. granulosus strains other than the G1 genotype.     

Molecular Characterization of Echinococcus granulosus Cysts in North Indian Patients: Identification of G1, G3, G5 and G6 Genotypes

Background

Cystic echinococcosis (CE) caused by the Echinococcus granulosus, is a major public health problem worldwide, including India. The different genotypes of E. granulosus responsible for human hydatidosis have been reported from endemic areas throughout the world. However, the genetic characterization of E. granulosus infecting the human population in India is lacking. The aim of study was to ascertain the genotype(s) of the parasite responsible for human hydatidosis in North India.

Methodology/Principal Findings

To study the transmission patterns of E. granulosus, genotypic analysis was performed on hydatid cysts obtained from 32 cystic echinococcosis (CE) patients residing in 7 different states of North India. Mitochondrial cytochrome c oxidase subunit1 (cox1) sequencing was done for molecular identification of the isolates. Most of the CE patients (30/32) were found to be infected with hydatid cyst of either G3 (53.1%) or G1 (40.62%) genotype and one each of G5 (cattle strain) and G6 (camel strain) genotype.

Conclusions/Significance

These findings demonstrate the zoonotic potential of G1 (sheep strain) and G3 (buffalo strain) genotypes of E. granulosus as these emerged as predominant genotypes infecting the humans in India. In addition to this, the present study reports the first human CE case infected with G5 genotype (cattle strain) in an Asian country and presence of G6 genotype (camel strain) in India. The results may have important implications in the planning of control strategies for human hydatidosis.     

中国北方汉族人群肌型肌酸激酶基因（CKMM）A/G多态研究

为研究中国汉族群体CKMM基因NcoI 酶切位点的遗传多态性以及该位点的具体多态形式, 采用PCR-RFLP技术, 对306例无血缘关系的健康中国北方汉人的染色体进行检测,并对3种基因型的扩增产物进行基因测序。用卡方检验对所得等位基因频率、基因型频率与其他种族进行比较。结果NcoI 位点多态性测序结果为：A/G颠换; 等位基因频率是A=86%,G=14%;基因型频率为：A/A=74%, A/G=24% , G/G=2%;经卡方检验符合Hardy－Weinberg遗传平衡定律;认为中国汉族群体CKMM 基因NcoI酶切位点具有遗传多态性。其基因型频率和等位基因频率在男女间没有显著性差异,与欧美人群相比有极显著差异,而与韩国人相比没有显著性差异。     

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.     

Genetic Diversity of Echinococcus granulosus in Center of Iran

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.