Molecular Characteristics and Serodiagnostic Potential of Dihydrofolate Reductase from Echinococcus granulosus

Abstract

The larval stage of Echinococcus granulosus causes cystic echinococcosis (CE), a neglected tropical disease that leads to morbidity and mortality in humans and livestock worldwide. Here, we identified and characterized dihydrofolate reductase (Eg-DHFR) from E. granulosus, and evaluated its potential as a diagnostic antigen for sheep CE. Comparison between mammalian (host) DHFR and Eg-DHFR indicates that 45.7% of the 35 active site residues are different. Immunolocalisation analysis showed that native Eg-DHFR was widely distributed in all life-cycle stages of E. granulosus. Recombinant Eg-DHFR (rEg-DHFR) showed typical DHFR enzymatic parameters towards substrate, and was very sensitive to inhibition by methotrexate (IC50 = 27.75 ± 1.03 nM) and aminopterin (IC50 = 63.67 ± 6.76 nM). However, inhibition of DHFR exhibited little protoscolicidal effect in vitro. As there is no reliable method to monitor sheep CE, the immunogenicity of rEg-DHFR was detected, and we developed an indirect ELISA (iELISA) for CE serodiagnosis. The iELISA exhibited diagnostic specificity of 89.58%, diagnostic sensitivity of 95.83%, and the diagnostic accuracy was 91.67% compared with necropsy. Cross-reactivity assay showed analytical specificity of 85.7%. These suggest that rEg-DHFR is an effective antigen for the diagnosis of sheep CE.

Preliminary Evaluation of Recombinant EPC1 and TPx for Serological Diagnosis of Animal Cystic Echinococcosis

“…Dihydrofolate reductases (DHFRs) are ubiquitous enzymes vital for purine and thymidylate synthesis in cells (Jennewein and Zmasek, 2013). In previous study, Song et al (2017) identified the serological diagnostic potential of recombinant DHFR, which had specificity of 89.58%, diagnostic sensitivity of 95.83%, and diagnostic accuracy of 91.67% in iELISA detection. Here, recombinant EPC1 exhibited poor specificity (49.4%) in detection of CE in sheep, indicating that rEPC1 is not suitable for ovine CE immunodiagnosis.…”

“…In addition, the protein with the best serodiagnostic potential was tested with the anti-Eg95 antibody. Furthermore, the tested antigens were compared with hydatid cyst fluid (HF), two previously reported recombinant immunogenic proteins (dihydrofolate reductase [rEg-DHFR] and rEg-P29) (Nouir et al, 2009;Song et al, 2017), and two commercial CE detection kits available in China. The study aimed to provide a sensitive and accurate detection method for CE to contribute to animal CE control.…”

Molecular and biochemical characterization of calmodulin from Echinococcus granulosus

“…Echinococcus granulosus protoscoleces (PSCs) and cyst walls were isolated aseptically using a previously reported method from liver hydatid cysts of naturally infected cattle presented for routine slaughter in an abattoir in Qinghai Province, China [ 14 ]. Two thousand PSCs were cultured in 1 ml of Roswell Park Memorial Institute (RPMI) 1640 medium with 10% bovine serum albumin (BSA; Hyclone, Logan, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, USA).…”

Modelling diagnostics for Echinococcus granulosus surveillance in sheep using Latent Class Analysis: Argentina as a case study

“…These techniques can be used to detect host antibodies specific to E. granulosus in serum samples, allowing for the detection of exposed/infected flocks without the need for necropsy. However, despite improvements to these approaches through the use of recombinant antigens, their diagnostic performance can be highly variable in the field, limiting their effectiveness as surveillance tools [ 23 , [25] , [26] , [27] , [28] ].…”