Collection of blood and faecal samples

Blood and faecal samples were obtained from 200 sheep brought to be slaughtered at a local abattoir in Giza, Egypt from January to June 2011. Based on gross inspection and post-mortem examination of the livers of the sheep after slaughter and subsequent parasitological examination of stool samples using the direct smear method, merthiolate–iodine–formaldehyde concentration method (Sapero & Lawless, Reference Sapero and Lawless1953) and Kato–Katz concentration technique (Siongok et al., Reference Siongok, Mahmoud, Guma, Warren, Muller, Handa and Houser1976), sheep were classified into three groups, namely 100 sheep infected with Echinococcus, 50 infected with Fasciola gigantica, Ascaris vitulorum or Strongyloides papillosus and 50 uninfected controls. Blood from each sheep was collected, centrifuged at 760 × g at 4°C for 10 min and serum samples were stored at − 20°C until analysed.

Forty E. granulosus infected human patients from areas in Dakahlia Governorate, where the disease is highly endemic, were admitted to tropical and surgical departments of Zagazig and El-Azhar University hospitals and were diagnosed, by means of sonography, computed tomography (CT) and magnetic resonance (MR) imaging as having CE in their livers. In addition, 30 patients infected with other parasites (Schistosoma mansoni or F. gigantica) were included. In addition, 30 members of the medical staff at Theodor Bilharz Research Institute served as parasite-free, healthy, negative controls. Blood samples were collected from all cases and sera were separated, aliquoted and kept at − 70°C until used.

Preparation of protoscolex antigen and polyclonal antibodies

Protoscoleces were prepared using the method of Rafiei & Craig (Reference Rafiei and Craig2002). In brief, protoscoleces were collected from fresh, fertile, sheep liver cysts. Viability was determined by the vital colouration approach, with 0.2% eosin staining. Protoscoleces were subjected to three cycles of freezing and thawing, washed three times, each time by centrifugation (600 × g for 5 min), and suspended in ten times their volume of 0.15 m phosphate-buffered saline (PBS) (pH 7.2). Subsequently, protoscoleces were suspended in four times their volume of PBS containing 0.1 mg aprotinin/ml, then sonicated on ice in a 150 W ultrasonic disintegrator, in 10-s bursts with 5-s intervals, until no intact protoscoleces were visible microscopically. The sonicate was left standing on ice for 1 h, then sedimented at 10,000 × g for 30 min. The supernatant was concentrated using an Amicon 8400 ultrafiltration unit with membrane (3 kDa cut-off) (EMD Millipore Corporation, Bedford, Massachusetts, USA). The sample was applied to DEAE-sephadex A50 column (ion exchange column chromatography) followed by a Sephacryl S-200 HR column (gel filtration chromatography) equilibrated in 0.1 m Tris–HCl, pH 7. The purity of the produced antigen was assayed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Laemmli, Reference Laemmli1970).

Two New Zealand white 4-month-old rabbits, weighing approximately 2 kg each, were injected intramuscularly with an equal volume of 1 mg protoscolex antigen (PA) and complete Freund's adjuvant (priming dose). Two weeks later they were injected with an equal volume of 0.5 mg PA and incomplete Freund's adjuvant (booster dose) and the latter booster dose was repeated twice, with a 1-week interval. Sera were collected from an ear vein 4 days after the last injection, to detect the titre of antibodies produced. When the titre was high, the animal was killed and exsanguinated and the sera were separated and stored at − 20°C until used (Fagbemi et al., Reference Fagbemi, Obarisiagbon and Mbuh1995; Guohadia & Fagbemi, 1997).

Purification, labelling and identification of the polyclonal antibody

The produced IgG polyclonal antibody (pAb) was purified using 50% ammonium sulphate precipitation (Nowotny, 1979) and caprylic acid treatment (McKinney & Parkinson, Reference McKinney and Parkinson1987). The protein content was estimated by a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, California, USA) (Bradford, Reference Bradford1976) and the purity of the produced IgG was identified by SDS-PAGE according to Laemmli (Reference Laemmli1970). The IgG pAb was conjugated with horseradish peroxidase according to the periodate method of Tijssen & Kurstak (Reference Tijssen and Kurstak1984). The reactivity of anti-protoscolex antigen IgG against Echinococcus antigens was assessed using the indirect ELISA (Engval & Perlman, Reference Engvall and Perlman1971).

Identification and characterization of the chemical nature of anti-PA IgG was performed using indirect ELISA by testing its reactivity against the antigen before and after treatment with 20 mm sodium periodate (Sigma Aldrich, Gillingham, Dorset, UK) and 4% trichloroacetic acid (Sigma Aldrich) (Woodward et al., Reference Woodward, Young and Bloodgood1985).

Detection of anti-protoscolex antigen IgG in sera

This method was performed, with some modifications from the original method of Engvall & Perlman (Reference Engvall and Perlman1971). Briefly, wells of the microtitration plates (Thomas Scientific, Swedesboro, New Jersey, USA) were coated with 100 μl of purified 27.5 kDa PA (10 μg/ml in 0.1 m carbonate buffer pH 9.6) and incubated overnight at room temperature. Plates were washed thoroughly three times with washing buffer (0.1 m PBS pH 7.4), then blocked with 200 μl of 0.1% bovine serum albumin (BSA) (Sigma) in 0.1 m PBS pH 7.4 for 1 h at 37°C. Plates were washed five times with washing buffer, then 100 μl of serum samples (1:250 diluted in washing buffer) were added to each well and incubated for 1 h at 37°C. Plates were washed three times with washing buffer and 100 μl of either polyvalent-anti-sheep (1:1000) or anti-human (1:2000) peroxidase conjugate was added to plates and incubated at room temperature for 30 min. The enzyme substrate reaction was stopped by adding 50 μl/well of 8 n H₂SO₄. The absorbance was measured at 492 nm using an ELISA reader (Bio-Rad Microplate Reader) (Allam et al., Reference Allam, Bauomy, Hemyeda, Diab and Sakran2012).

Detection of circulating protoscolex antigen in sera

After several optimization trials, the following sandwich ELISA was performed for circulating protoscolex antigen (CPA) detection, using anti-27.5 PA IgG pAb as coating and peroxidase-conjugated antibody for capture of the antigen. For each step, 100 μl/well was added unless mentioned otherwise. Microtitration plates were coated with the anti-27.5 PA IgG pAb (20 μg/ml) in 0.1 m carbonate buffer, pH 9.6 and left overnight at room temperature. Plates were washed thoroughly three times with phosphate-buffered saline/Tween (PBS/T) 2 min each wash, and unbound sites were blocked with 200 μl/well of 2.5% fetal calf serum (FCS)/PBS/T and incubated for 2 h at 37°C. Following emptying the plates by suction, undiluted serum was added and incubated for 2 h at 37°C. After thorough washing as described above, peroxidase-conjugated anti-27.5 PA IgG pAb (1:1000 diluted in PBS/T) was added. Plates were incubated for 1 h at 37°C and washed with PBS/T. The substrate O-phenylenediamine dihydrochloride (Sigma) was added and the plates were incubated for 30 min in the dark at room temperature. The enzyme reaction was stopped with 50 μl/well of 8 n H₂SO₄. The absorbance was measured at 492 nm using an ELISA reader (Bio-Rad). The cut-off value was calculated as the mean optical density (OD) reading of negative controls + two standard deviations of the mean. The OD readings equal to or less than the cut-off value were considered negative, while those readings greater than the cut-off value were considered positive.

The specificity of the assays was determined as the sum of the data from the negative controls and the group of sheep or humans infected with other parasites.

Data analysis

Data were expressed as mean ±  standard deviation and analysed using the SPSS computer program (Version 16 Windows; SPSS Inc., Chicago, Illinois, USA). Standard diagnostic indices were calculated as described by Galen (Reference Galen1980).