Abstract. A standardized test for the serodiagnosis of cystic echinococcosis (CE) remains an important challenge because of the problems in specificity and sensitivity of the available commercial kits and lack of proper evaluation of antigen. Using appropriate sources of antigenic material is crucial in improvement of the serological methods such as enzyme-linked immunosorbent assay (ELISA). This study was conducted to evaluate the performance of protein named Echinococcus protoscolex calcium binding protein EPC1 for the detection of antibodies in sera from patients with CE. Expressed and purified recombinant protein EPC1 (rEPC1) was used as antigen in ELISA method. Characterization of the rEPC1 antigen was evaluated using the serum of 25 patients with both surgical and imaging confirmed CE and 25 healthy donors as negative controls. Also, a panel of sera including chronic toxoplasmosis (IgG positive), strongyloidosis, fascioliasis, toxocariasis, and kala azar were used and patients with related parasites were confirmed by medical laboratories or clinically by research centers using microscopy or specific ELISA. rEPC1 showed relatively promising performance in total IgG ELISA for the detection of antibodies in sera from the negative controls, and the cut off value 0.4 units of optical density at 490 nm was calculated for ELISA. In this study, sensitivity of 100%, specificity of 93.7, positive predictive value of 92.6%, and negative predictive value of 100% were calculated for rEPC1. On the other hand, commercial ELISA kit based on the native antigen B of Echinococcus granulosus had sensitivity of 96.2% and specificity of 96.8%. No significant difference was found for sensitivity or specificity between the rEPC1 and commercial kit. However, rEPC1 may be a valuable antigen for diagnosis of human CE.
It is important to establish the diagnosis of cystic echinococcosis (CE) infection and begin control management. Currently, it is difficult to make an accurate diagnosis of CE without the availability of an accurate test, which requires the use of sensitive and specific antigens. Using recombinant antigens the sensitivity and specificity of the CE serology assays could be improved considerably. Recently, a highly antigenic protein named EPC1was characterized and isolated from an Echinococcus granulosus protoscoleces. The current study was designed to assess the sequences of EPC1 isolated from different intermediate hosts of E. granulosus. In addition, identification of a highly antigenic linear B cell epitope was found within EPC1 antigen candidate. The EPC1 sequence contains coding and non-coding regions and was compared between two predominant strains (G1 and G6) in Iran. Sequence polymorphism was not found in protein coding regions, suggesting that these regions may be useful for identification of protein expression as an antigen. The average antigenic activity for the whole protein is above 1.1, and hydrophobicity below 0 indicates that it is hydrophilic. Structural analysis showed alpha helical regions in amino acids 6-25, 35-44, 52-62, and 72-78. Nine B cell epitope residues were identified out of 67 total residues. The identity of EPC1 sequence in both G1 and G6 genotypes affects the antigenic efficacy of EPC1and suggests the recombinant protein will be useful in serological assays in the regions where the two strains are prevalent.
Background
Genomic comparison of Mycoplasma synoviae vaccine strain MS-H and the MS-H parental strain 86,079/7NS established a preliminary profile of genes related to attenuation of MS-H. In this study we aimed to identify the stability of mutations found in MS-H after passage in experimental or field chickens, and to evaluate if any reverse mutation may be associated with changes in characteristics of MS-H in vitro or in vivo.
Results
Whole genome sequence analysis of 5 selected MS-H field reisolates revealed that out of 32 mutations reported previously in MS-H, 28 remained stable, while four found to be reversible to the wild-type. Each isolate possessed mutations in one to three of the genes obg, oppF1 and gap and/or a non-coding region. Examination of the 4 reversible mutations by protein modeling predicted that only two of them (in obg and oppF1 genes) could potentially restore the function of the respective protein to that of the wild-type.
Conclusions
These results suggest that the majority of the MS-H mutations are stable after passage in vaccinated chickens. Characterisation of stable mutations found in MS-H could be utilised to develop rapid diagnostic techniques for differentiation of vaccine from field strains or ts- MS-H reisolates.
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