Detection of Echinococcus granulosus sensu lato infection by using extracts derived from a protoscoleces G1 cell line

Abstract

Cystic echinococcosis (CE), also called hydatidosis, is a zoonotic parasitosis worldwide distributed 1 caused by the larval stage (metacestode) of Echinococcus granulosus sensu lato. This hermaphrodite worm develops in the intestine of domestic and wild canids, and then its gravid proglottid releases the eggs to the faeces, contaminating the environment. The eggs ingested by the intermediate host hatch in the duodenum, and the oncosphere then penetrates the intestinal wall, migrates, and becomes trapped in the host's organs, where it becomes metacestode. The parasite's cycle is accomplished when the infected organ from the intermediate host is ingested by the definitive host (canids). 2 Humans, as intermediate accidental hosts, who are more exposed to the infection in unsanitary conditions, develop a parasitic disease involving the encystment of the larvae in different organs (the liver in 70%-80% of the cases, the lungs in 20%-30% of the cases and other organs with lower frequency), 3 resulting in a wide spectrum of clinical manifestations. Mitochondrial DNA has so far allowed identifying eight Echinococcus granulosus sensu lato genotypes (G1, G3-G8, and G10). 4,5 Four of these genotypes, G1 in sheep, G5 in cattle, G6 in camelids and G7 in swine, have been detected in Argentina. 4 Abstract Cystic echinococcosis (CE) can be diagnosed by means of several serological approaches, but their results vary among laboratories due to the molecular characteristics of the reference antigens used. Thus, this study aimed to address both the relevance of an EGPE cell line previously obtained from Echinococcus granulosus protoscoleces G1 and the complexity of the immune response by using two different in vitro growth stages as separate sources of parasite antigens. The serum reactivity was investigated by western blotting (WB) in 21 CE patients from an endemic area in a matched case-control design and also in seven experimentally infected sheep and five healthy control sheep. EGPE-antigen-human serum sensitivity by WB was higher than that of hydatid fluid (HF) WB, ELISA and DD5 (P < .05, Chi-square test). EGPE protein extract was immunogenic in mice and hyperimmune plasma reacted with HF proteins, and AgB2 expression was detected by molecular analysis. Proteins of 37 to 60 kDa were recognized by 95.24% of the CE patients' sera but, with poor specificity. Statistically significant differences were found between serum protein extract recognition at 7 and 20 days of cell growth. The EGPE cell line is a laboratory source of antigens for improvement of CE serological diagnosis. K E Y W O R D S cell line extract, Cystic echinococcosis, human, immunoblot, parasite, sera, sheep

Characterization of the B-Cell Epitopes of Echinococcus granulosus Histones H4 and H2A Recognized by Sera From Patients With Liver Cysts

“…All protocols and procedures were approved by the Ethics Committee of the ‘Universidad Abierta Interamericana’, Buenos Aires, Argentina (number 01011). Patient serum samples were from an anonymized laboratory serum stock ( Maglioco et al., 2019 ).…”

“…Briefly, EGPE cells were grown in medium 199 (Sigma), 1 mM sodium pyruvate (sodium salt, extra pure, Anhedra, Beijing, China) and 78 μg/mL β-mercaptoethanol (Merck, Darmstadt, Germany) at pH 7.9 (37°C; CO 2 :air; 5/95%) and EGPE cell colonies were performed in 2% agarose (20,000 cells/well) ( Echeverría et al., 2010 ). Cells were grown in a liquid medium for 20 days and protein extracts were obtained as previously ( Maglioco et al., 2019 ). Briefly, cells were washed five times with DPBS and incubated in lysis buffer (8 mmol/L CHAPS,MP Biomedicals, 10 mmol/L Tris –HCl, pH 8, 2 mmol/L EDTA, 0.1% B‐mercaptoethanol,MP Biomedicals, and 1/100 protease inhibitor cocktail, Sigma‐Aldrich), at 4°C for 2 hours.…”

“…Every fraction with higher absorbance was concentrated through a 3K cut‐off membrane concentrator (Pierce, Thermo Scientific). The reactivity of these fractions and that of the supernatant of EGPE colonies was analyzed by Western blot ( Maglioco et al., 2019 ) by using a pool of sera from 11 patients with CE with only hepatic localization ( Supplementary Figure 1 ). Then, reactive protein fractions were passed through an affinity column (Protein G HP SpinTrap - GE Healthcare) prepared with this pool of CE sera (cases) or through an affinity column performed with a pool of sera from two patients with cysticercosis and two patients with fascioliasis (controls).…”

“…In our laboratory, a cell line called EGPE was obtained from E. granulosus sensu lato G1 strain from bovine liver ( Echeverría et al., 2010 ). By using this cell line in a paired case-control study, we have previously found that serum from CE patients recognizes protein extracts from EGPE cells at two growth stages with higher sensitivity than those extracted from hydatid fluid ( Maglioco et al., 2019 ).…”