Characterization of the eg95 gene family in the G6 genotype of Echinococcus granulosus

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Abstract

Cystic echinococcosis in humans and livestock animals is caused by infection with the cestode parasite Echinococcus granulosus. A number of genotypes of the parasite (designated G1–G10) are known to exist, with the genotype cluster G1–G3 and genotype G6 being responsible for the majority of humans infections. A recombinant vaccine has been developed for use in livestock to prevent infection with E. granulosus. The vaccine is based on the antigen EG95 which is expressed in the early larval stage (oncosphere) of the parasite. The EG95 antigen was originally cloned from the G1 genotype of E. granulosus and the protein has been found to be encoded by members of a small family of related genes in this genotype. Reliable information has not been available about the likely efficacy of the EG95 vaccine against genotypes other than G1. In this study, genomic DNA cloning techniques were used to characterize seven eg95-related gene fragments from the G6 genotype of E. granulosus. Three proteins appear to be encoded by these genes. Considerable differences were found between the EG95 related proteins from the G6 genotype compared with the EG95 protein from the G1 genotype. These differences suggest that the EG95-related proteins from the G6 genotype may have different antigenic epitopes compared with the current vaccine antigen. Data presented in this study have implications for future vaccine design and provide the information that would enable a G6 genotype-specific vaccine to be developed against E. granulosus, should this be considered a desirable addition to the available tools for control of cystic echinococcosis transmission.

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.

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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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    Note: Nucleotide sequence data reported in this paper are available as GenBank ID: JQ285934, JQ285935, JQ285936, JQ285937, JQ285938, JQ285939 and JQ285940.

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