Characterization of the eg95 gene family in the G6 genotype of Echinococcus granulosus☆
Graphical abstract
Seven eg95-related genes were found in the G6 genotype. They encode three proteins showing considerable differences with the EG95 vaccine from G1.
Highlights
► There are at least seven eg95-related genes in the genotype of the G6 genotype of Echinococcus granulosus. ► These genes appear to encode three proteins. ► These proteins show considerable differences with the EG95 vaccine from G1. ► EG95 proteins from G6 may have different antigenic epitopes compared with the current vaccine. ► Data presented provide the information for the development of a G6 genotype-specific vaccine.
Introduction
Echinococcus granulosus causes cystic echinococcosis in animals and humans. The parasite has a worldwide distribution with the highest prevalence occurring in parts of Eurasia, north and east Africa, and South America [1]. The disease has re-emerged in some areas and is still present in many areas where control programs have been implemented [2]. Genetic variability has long been recognized in E. granulosus with a number of genotypes described and designated G1–G10 [3], [4], [5]. The taxonomy of the genus has been reviewed and several genotypes of what were previously considered to be variants of E. granulosus are now considered to be regarded as distinct species [6], [7]. The G1–G3 cluster genotype is the most common and is responsible for most human infection [8], [9], [10], [11], [12]. The G6 genotype is also an important aetiological agent of human cases in specific areas [13], [14], [15].
A vaccine has been developed for use against E. granulosus infections in livestock intermediate hosts so as to reduce transmission of the parasite and indirectly reduce the incidence of infection in humans [16]. The vaccine utilizes a recombinant protein, designated EG95, which is uniquely expressed in the parasite's oncosphere life cycle stage [17]. EG95 has been found to induce high levels of protection (96–100%) in experimental vaccine trials undertaken in sheep and other intermediate hosts in a number of countries against challenge infections with E. granulosus either known or believed to be of the G1 genotype [18], [19], [20]. Investigations by Chow et al. [21] found that the EG95 antigen was encoded by members of a family of genes in the G1 genotype, with four genes encoding the same EG95 protein antigen, while two other genes were found to encode related proteins and another was predicted to be a pseudogene. Currently an E. granulosus G1 genome sequencing program is being undertaken at the Wellcome Trust Sanger Institute led by Matt Berriman in collabo-ration with Cecilia Fernandez (Universidad de la Republica, Uruguay), however the available dataset (December 2011) [22] appear to be incomplete and do not yet provide accurate information about the number of eg95-related genes in the genome [23]. It was recognized from the earliest times in the development of the EG95 vaccine that there was a need to characterize EG95-related proteins in different isolates and genotypes of E. granulosus in order to assess the vaccine's potential for protection against different E. granulosus strains [24]. Subsequently, investigations into the variability of eg95-related genes have been undertaken by a number of groups using PCR-based strategies with non gene-specific primers [25], [26]. One of these studies in particular revealed a high degree of variability in the eg95 gene family members [25]. However the reliability of the data obtained is unclear because some of the amplified products could be the results of a number of different artifacts that are known to occur when PCR is used with non-gene specific primers [27]. A preliminary study of eg95-related genes in E. granulosus G6/G7 was performed by Chow et al. [28] using gene specific primers in PCR. A single eg95-related gene was identified from both G6 and G7 genotypes, showing substantial nucleotide variability with the eg95 gene family members from G1. In the absence of complete and reliable data about eg95-related genes in the G6 genotype of E. granulosus, full characterization of eg95-related genes from this genotype was undertaken in this study.
Section snippets
Extraction of parasite nucleic acids and Southern blot experiments
Fresh protoscoleces were collected from individual hydatid cysts from naturally infected camels in slaughterhouses in Iran. DNA was extracted as previously described [29]. Following phenol/chloroform extraction, total nucleic acids were precipitated with isopropanol and resuspended in sterile distilled water and stored at −20 °C. The parasite genotype was determined using PCR with genomic DNA as template and amplifying 366 bp from the cox1 gene [3]. Genomic DNA was digested with EcoRI or XhoI,
Southern blot
Genomic DNA identified as belonging to the G6 strain of E. granulosus and digested with EcoRI showed a hybridization pattern consisting of six bands (designated I to VI, Fig. 1). The bands ranged from 11 to 1.2 kb in size and hybridized with the full length eg95 cDNA probe. Hybridization of the digested G6 genomic DNA with the 5′ eg95 cDNA probe showed a pattern containing bands II, III, V and VI (Fig. 1, panel B). Hybridization with the 3′ probe showed a pattern with bands I and IV as shown in
Discussion
Seven eg95-related gene fragments were identified from the G6 genotype of E. granulosus all of which contained an internal EcoRI restriction site (Fig. 1A–C). None of the sequences identified from the G6 genotype was identical to the genes encoding the EG95 vaccine antigen from G1 [21], as shown in Table 1. Genes eg95-1G6 and eg95-2G6 show the highest similarity to eg95-1G1 (97%). Nucleotide sequence alignments and comparisons to the eg95-1G1 gene suggest that eg95 genes from G6 possess a
Acknowledgements
Cristian A. Alvarez Rojas is a recipient of a MRS and MIRFS scholarships from The University of Melbourne. Funding is acknowledged from Australia National Health and Medicine Council, grant numbers 350279, 628320, 100354.
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2018, Computational Biology and ChemistryCitation Excerpt :The in silico-based MPMVs that are assembled from the B- and T-cell epitopes of important vaccine candidate antigens (VCAs) may be employed as a promising vaccination platform to interfere with and hinder the parasite biological cycle (Pourseif et al., 2017). Many VCAs have so far been served for development of different types of recombinant vaccines against E. granulosus (Li et al., 2014; Alvarez Rojas et al., 2012; Hui et al., 2009; Zhou and Li, 2008; Petavy et al., 2008). In this regard, immunization with EG95-based vaccines has been examined with different hosts and different infectious challenges.
Genetic diversity and phylogenetic analysis of EG95 sequences of Echinococcus granulosus: Implications for EG95 vaccine application
2017, Asian Pacific Journal of Tropical MedicineCitation Excerpt :This might allow parasites escaping the anti-EG95 immune responses by expressing this protein, which made the parasite insusceptible to the immune responses induced by the current EG95 vaccine. Although the EG95 gene family has been characterized in G1 and G6 strains [12,16], the validity of their results might be limited due to small number of EG95 isolates. In contrast, this study covered all published EG95 sequences in GenBank and the results might be more comprehensive.
Echinococcus granulosus sensu lato genotypes infecting humans - review of current knowledge
2014, International Journal for ParasitologyCitation Excerpt :The EG95 protein has been found to be encoded by members of a small family of genes (Chow et al., 2001). Recent investigation of the genes encoding EG95-related proteins in the G6 genotype (Alvarez Rojas et al., 2012) identified differences in the predicted amino acid sequence of the proteins from those described from the G1 genotype. The protein in G6 most closely related to the EG95 vaccine protein from G1 was found to differ from it at seven amino acid positions.
Maternal antibody parameters of cattle and calves receiving EG95 vaccine to protect against Echinococcus granulosus
2012, VaccineCitation Excerpt :The pig adapted genotype, Echinococcus canadensis (G6/7) is commonly found in cattle in some regions of the world and may even be the dominating species causing human infections in certain areas [36] although in global terms it is a minor contributor to human disease compared to E. granulosus sensu stricto. Recent evidence indicates that the homologues of EG95 are different in E. canadensis [37] and that there will need to be further studies to determine if the current vaccine would protect against infection with E. canadensis or whether a species-specific vaccine would be required [38]. With respect to the studies described here using the G1 genotype (E. granulosus sensu stricto), this is clearly the most important genotype infecting humans worldwide and the current vaccine provides excellent protection which can largely prevent cattle from being capable of contributing to transmission of the parasite.