Human and camel cystic echinococcosis – a polyclonal antibody-based sandwich ELISA for its serodiagnosis with molecular identification

Veterinary Research Communications https://doi.org/10.1007/s11259-024-10375-3 RESEARCH Human and camel cystic echinococcosis – a polyclonal antibody-based sandwich ELISA for its serodiagnosis with molecular identification A. Maher1 · N. I. Toaleb2 · R. M. Shaapan1 · D. Aboelsoued2 · M. B. Salman1 · S. Zaky3 Received: 16 December 2023 / Accepted: 4 April 2024 © The Author(s) 2024 Abstract Cystic echinococcosis (CE) is an emergent neglected disease affecting human and animals in Egypt with a wide distribution and incidence. This study aimed to evaluate the use of a polyclonal antibody-based sandwich ELISA in the detection of Echinococcus granulosus antigen in human and camel sera. Hydatid cyst protoscoleces antigen (PsAg) was isolated from hydatid cysts collected from naturally infected camel livers and lungs. PsAg was used for immunization of rabbits to raise IgG polyclonal antibodies (IgG PsAb). IgG PsAb were then precipitated, purified using Protein-A Sepharose gel and labeled with horseradish peroxidase enzyme. We assayed the purity of the IgG PsAb, and the two prepared E. granulosus antigens CPsAg from camel cysts and HPsAg from human cysts by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The resulted protein bands of the prepared CPsAg appeared at different molecular weights: 180, 90, 68, 54, 42 and 22 kDa while, HPsAg shared with it in 4 common bands at 68, 54, 42, and 22 kDa. The purified IgG PsAb had been resolved at two bands at 52 kDa and at 32 kDa. Sandwich ELISA were performed for the detection of circulating E. granulosus antigens in sera of human (n = 183) and camels (n = 190). The purified IgG PsAb showed strong reactivity against E. granulosus infected human and camel samples and no cross reactivity neither with free-healthy negative sera nor with others parasitic diseases (Schistosomiasis, Fascioliasis, Toxoplasmosis, Ancylostomiasis for human samples and Fascioliasis, ticks’ infestation, Eimeriosis, Cryptosporidiosis, Nasal myiasis, Toxoplasmosis for camel samples). The sensitivity of the assay was 98.25% (56/57) and 96.9% (31/32) against human and camel samples, respectively. Specificity was 100% in both human and camel samples. Sandwich ELISA detected CE in 33.3% (24/72) and 55.6% (50/90) random human and camel samples, respectively. Indirect ELISA, using CPsAg, was used for detection of antibodies in positive human and camels’ sera and detected 96.5% (55/57) and 93.8% (30/32) of human and camel samples, respectively. In our study, Genomic DNA was extracted from protoscoleces fluid of human liver hydatid cysts to identify the Echinococcus sp. isolate based on NADH dehydrogenase subunit 1 (NAD1) gene by Polymerase Chain Reaction (PCR) and the isolate (GenBank: OP785689.1) were identified as E. granulosus sensu lato genotype. In conclusion, Sandwich ELISA technique was found to be a potent and sensitive assay for detection of hydatid antigen in both human and camel samples. Keywords Echinococcus granulosus · PCR · Protein-A sepharose gel affinity chromatography · SDS-page · Sandwich ELISA · Indirect ELISA D. Aboelsoued M. B. Salman S. Zaky 1 A. Maher Department of Zoonotic Diseases, Veterinary Research Institute, National Research Centre, Dokki, Giza, Egypt 2 Department of Parasitology and Animal Diseases, Veterinary Research Institute, National Research Centre, Dokki, Giza, Egypt 3 Hepato-Gastroenterology and Infectious Diseases Department, Faculty of Medicine, Al-Azhar University, Cairo, Egypt N. I. Toaleb R. M. Shaapan 13 Veterinary Research Communications Introduction Cystic echinococcosis (CE) is important helminthic zoonotic disease caused by the infection with Echinococcus granulosus belonging to the family Taeniidae (Thompson 2017; Ebrahimipour et al. 2019; Zhang et al. 2023). Fecal materials of E. granulosus-infected dogs and canids (definitive hosts) contaminate the environment spreading infective eggs which are taken by fecal-oral route in humans and animals (intermediate hosts) (King and Fairley 2015) causing significant morbidity and mortality in humans as well as significant economic losses in livestock industry worldwide (Nigo et al. 2022; Cai et al. 2023). In livestock, CE could result in reduced birth rate, low yield and quality of animal meat and wool, delayed growth, destructed viscera, organ condemnation, and even death (Benner et al. 2010; Cai et al. 2023). In humans, economic losses are resulted from increased surgery costs, hospital care, and impaired labor productivity (Singh et al. 2014; Sen et al. 2019). Its prognosis depends on cyst number, stage, and location making control of this disease complex (Ali et al. 2020). The high prevalence of CE in humans and animals is usually observed in temperate regions (Mediterranean regions, North and East Africa, and Central Asia) (Grosso et al. 2012). Cysts are fluid-filled vesicles with two layers. The outermost laminated layer which is an acellular coat secreted by the inner germinal layer. The inner germinal layer is the cellular proliferative sheet of the hydatid cyst which encloses brood capsules, protoscoleces (Pedrosa et al. 2000), undifferentiated cells, storage cells, and muscle cells (Siles-Lucas et al. 2017). Due to absence of specific clinical signs and symptoms, CE is commonly performed at necropsy in postmortem (PM) examination in animals (Craig et al. 2015) and by laboratory techniques and radiological imaging in humans (Alli et al. 2011). Ultrasonography, computerized tomography, and serology are useful diagnostic tools for human hydatidosis, and the disease is considered a public health challenge and needs accurate differential diagnosis from any cystic mass/s in the abdomen (Elaadli et al. 2022). Immunodiagnostic laboratory techniques are widely used to confirm CE diagnosis in suspected cases or follow up after surgical and chemical treatments, especially Enzyme Linked Immunosorbent Assay (ELISA) and Indirect hemagglutination test (IHAT) (Zhang et al. 2012). ELISA is a fast, easy and non-expensive test to perform with high sensitivity and specificity (Hassanain et al. 2021). The main obstacle of antibody tests is that they couldn’t distinguish between past and present infections. Antigen detection techniques might help to avoid this problem (Doiz et al. 2001) and might be useful in the immunodiagnosis of hydatid disease (Toaleb et al. 2023). Circulating hydatid antigens are detected in the serum only during active 13 infection, and their levels decrease gradually after surgical removal of the cyst or successful chemotherapy (Devi and Parija 2003; Sadjjadi et al. 2009; Bauomi et al. 2015). Also, antigen detection assays could help in assessing the efficacy of treatment, especially after surgical removal of hydatid cyst (Bauomi et al. 2015). Because of the importance of cystic echinococcosis in human and animal health, rapid and precise diagnostic methods are urgently needed (Hassanain et al. 2021). Consequently, th (...truncated)