BACKGROUNDEchinococcosis, coenurosis and cysticercosis are debilitating diseases which prevail in China. Immunological diagnosis of metacestodosis is important in disease control. The 8-kDa glycoproteins from taeniid cestodes have successfully been used for diagnosis of human cysticercosis in immunological assays. The aim of the present study was to investigate genetic variations and phylogenetic relationships of the 8-kDa proteins for evaluating the possibility of utilizing these proteins as diagnostic antigens for other metacestode infections.

METHODSThe genes and complementary DNAs (cDNAs) encoding the 8-kDa proteins from Echinococcus (E.) granulosus, Taenia (T.) multiceps and T. hydatigena were amplified using PCR method. Their amplicons were cloned into the vector pMD18 and the positive clones were sequenced. Sequence data were analyzed with the SeqMan program, and sequence homology searches were performed using the BLAST program. Alignments were conducted using the ClustalX program, and the phylogenetic analyses were performed with the Protein Sequences Program and the Puzzle Program using the Neighbor-joining method.

RESULTSFifteen, 18 and 22 different genomic DNA sequences were identified as members of the 8-kDa protein gene family from E. granulosus, T. multiceps and T. hydatigena, respectively. Eight, four and six different cDNA clones respectively from E. granulosus, T. multiceps and T. hydatigena were characterized. Analysis of these sequences revealed 54 unique 8-kDa protein sequences. Phylogenetic trees demonstrated that the taeniid 8-kDa proteins are clustered into eight clades at least: Ts18, Ts14, TsRS1, TsRS2, T8kDa-1, T8kDa-2, T8kDa-3 and T8kDa-4.

CONCLUSIONWe found that the gene family encoding for the taeniid 8-kDa antigens is comprised of many members with high diversity, which will provide molecular evidence for cross-reaction or specific reaction among metacestode infections and may contribute to the development of promising immunological methods for diagnosis of metacestodosis.

Amino Acid Sequence ; Animals ; DNA, Helminth ; genetics ; Echinococcus granulosus ; genetics ; metabolism ; Genetic Variation ; genetics ; Glycoproteins ; chemistry ; classification ; genetics ; Helminth Proteins ; chemistry ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Sequence Homology, Amino Acid ; Taenia ; genetics ; metabolism

The aim of the present study is to determine the characteristics of genotype and phenotype of Echinococcus granulosus derived from wild sheep and to compare them with the strains of E. granulosus sensu stricto (sheep-dog) and E. granulosus camel strain (camel-dog) in Iran. In Khojir National Park, near Tehran, Iran, a fertile hydatid cyst was recently found in the liver of a dead wild sheep (Ovis orientalis). The number of protoscolices (n=6,000) proved enough for an experimental infection in a dog. The characteristics of large and small hooks of metacestode were statistically determined as the sensu stricto strain but not the camel strain (P=0.5). To determine E. granulosus genotype, 20 adult worms of this type were collected from the infected dog. The second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) and cytochrome c oxidase 1 subunit (COX1) of the mitochondrial DNA were amplified from individual adult worm by PCR. Subsequently, the PCR product was sequenced by Sanger method. The lengths of ITS2 and COX1 sequences were 378 and 857 bp, respectively, for all the sequenced samples. The amplified DNA sequences from both ribosomal and mitochondrial genes were highly similar (99% and 98%, respectively) to that of the ovine strain in the GenBank database. The results of the present study indicate that the morpho-molecular features and characteristics of E. granulosus in the Iranian wild sheep are the same as those of the sheep-dog E. granulosus sensu stricto strain.
Animals ; DNA, Helminth/genetics ; DNA, Ribosomal Spacer/genetics ; Dogs ; Echinococcosis/parasitology/*veterinary ; Echinococcus granulosus/anatomy & histology/classification/genetics/*physiology ; Electron Transport Complex IV/genetics ; *Genotype ; Iran ; *Phenotype ; Phylogeny ; Polymorphism, Restriction Fragment Length ; Sheep ; Sheep Diseases/*parasitology ; Species Specificity

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.
Animals ; Cattle ; Cluster Analysis ; DNA, Helminth/chemistry/genetics ; Echinococcosis/parasitology/*veterinary ; Echinococcus granulosus/*classification/*genetics/isolation & purification ; Electron Transport Complex IV/genetics ; *Genetic Variation ; Genotype ; Goats ; Iran ; Phylogeny ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; Sheep

Echinococcus granulosus is the causative agent of cystic echinococcosis with medical and veterinary importance in China. Our main objective was to discuss the genotypes and genetic diversity of E. granulosus present in domestic animals and humans in western China. A total of 45 hydatid cyst samples were collected from sheep, humans, and a yak and subjected to an analysis of the sequences of mitochondrial cytochrome b (cytb) gene. The amplified PCR product for all samples was a 1,068 bp band. The phylogenetic analysis showed that all 45 samples were identified as E. granulosus (genotype G1). Ten haplotypes were detected among the samples, with the main haplotype being H1. The haplotype diversity was 0.626, while the nucleotide diversity was 0.001. These results suggested that genetic diversity was low among our samples collected from the west of China based on cytb gene analysis. These findings may provide more information on molecular characteristics of E. granulosus from this Chinese region.
Animals ; Animals, Domestic/parasitology ; Base Composition ; Base Sequence ; Cattle/*parasitology ; China ; Cytochromes b/*genetics ; DNA, Helminth/genetics ; Echinococcosis ; Echinococcus granulosus/classification/*genetics ; Genetic Variation ; Haplotypes/genetics ; Humans ; Mitochondria/genetics ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction/*veterinary ; Sequence Alignment ; Sequence Analysis, DNA ; Sheep/*parasitology ; Tibet