Three recombinant proteins of E. granulosus including two EgAgB sub-units EgAgB8/1 and EgAgB8/2 and Echinococcus protoscolex calcium binding protein (EPC1) were expressed in the prokaryotic expression vectors. Total RNA was isolated from the protoscolices retrieved from a fertile hydatid cyst from buffalo liver at a local abattoir using Trizol reagent (Invitrogen, USA). Briefly, ~0.2 x10⁴ protoscolices were treated with Trizol reagent (1ml) and manually homogenized with a micropestle in a sterile 2.0 ml microcentrifuge tube. The lysed protoscolices were freeze-thawed at -80°C for multiple cycles together with manual homogenization to completely lyse the parasites. Total RNA was isolated from the lysed protoscolices following standard RNA isolation protocol (Invitrogen, USA). The RNA was converted to single stranded cDNA using oligo-dT primer and reverse transcriptase enzyme (MBI Fermentas, USA) following standard protocols of cDNA synthesis. The cDNA coding for each of the above three target proteins was PCR amplified with gene specific primers (Table1). The PCR products of three target cDNAs were cloned in p^DRIVE cloning vector (Qiagen, Germany) and sequence confirmed for each cDNA.

Expression of the recombinant EgAgB8 sub-units and EPC1 protein
The cDNAs coding for EgAgB8/1, EgAgB8/2 and EPC1 proteins were PCR amplified with primers designed with suitable restriction enzyme sites and expressed in three different prokaryotic expression vectors (Table 1).

EgAgB8/1 protein was expressed in p^Rham vector in frame with solubility enhancing sumo-fusion protein (Expresso™ Rhamnose cloning and expression kit; Lucigen, USA). E. cloni® 10G chemically competent cells were transformed with the recombinant p^Rham vector and a moderate level of the recombinant fusion protein was generated on induction of the culture with 0.2% L-rhamnose for 8 h at 37^oC. The bacterial cells were disrupted with 6 M guanidine hydrochloride in the lysis buffer (pH 8.0) supplemented with 18 mM imidazole and 10 mM â-mercaptoethanol, followed by sonication of the cell lysate with 3 bursts of 30 sec each at 10 micron amplitude. The protein was purified to complete homogeneity using Ni-NTA affinity chromatography. The recombinant protein was bound to Ni-NTA resin (Qiagen, Germany) at room temperature for 2 h with constant shaking. Affinity column was washed with wash buffer (pH 6.0) supplemented with 18 mM imidazole and protein eluted with elution buffer (pH 4.2).

Expression of the EgAgB8/2 recombinant protein was carried out in p^{ET32a (+)} vector in Escherichia coli BL21 (DE3).The cDNA was PCR amplified and cloned in the expression vector in frame with the vector fusion tag. An optimum level of expression of the EgAgB8/2 recombinant protein was achieved at 6 h post-1mM IPTG induction at 37 ^oC. The recombinant protein was purified by lysis of the bacterial cells in lysis buffer (pH 8.0) containing 8 M urea and supplemented with 12 mM imidazole and 10 mM â-mercaptoethanol for 2 h at RT. The cell lysate was then sonicated on ice for 5 cycles of 30 sec each at 5 micron amplitude. The recombinant protein was bound to Ni-NTA resin at room temperature for 2 h with constant shaking. The affinity column was washed with wash buffer (pH 6.5) supplemented with 12 mM imidazole and recombinant protein eluted with elution buffer at pH 4.2.

The EPC1 target protein was expressed in p^PROEXHT-b vector, with a higher level of expression of the recombinant protein achieved at 8 h post-IPTG induction at 37^oC. E. coli BL21 (DE3) cells induced with 1mM IPTG were disrupted in lysis buffer (pH 8.0) containing 6 M guanidine hydrochloride and supplemented with 16 mM imidazole at room temperature for 2 h and the recombinant protein was purified by Ni-NTA affinity chromatography. The wash buffer (pH 5.9) was supplemented with 16 mM imidazole and recombinant protein was eluted at pH 4.2. The composition of the lysis, wash and elution buffers used in the purification steps of each recombinant protein was 10 mM tris and 100 mM potassium dihydrogen phosphate containing 6M guanidine hydrochloride or 8M urea as protein denaturant.

The purified recombinant fusion proteins EgAgB8/1, EgAgB8/2 and EPC1 resolved at 25, 29 and 10.5 kDa, respectively in the SDS-PAGE.

Collection of goat sera
Goats (n=116) were screened for hydatid infection at necropsy at the local abattoir. Out of the 116 animals examined at necropsy, 15 were positive for hydatid cysts in the liver or lungs and remaining 101 were negative for hydatid cysts. Sera were retrieved from these animals and screened for anti-hydatid antibodies by IgG-ELISA with the above three recombinant antigens. Sera were also collected from goats infected with other parasites for their cross-reactivity studies. Sera collected from healthy goats, maintained at Indian Veterinary Research Institute, Izatnagar, were used as negative control in the subsequent immuno-assays. All experiments on goats were conducted as per the guidelines of the Institute Animal Ethics Committee.

Enzyme Linked Immunosorbent Assay
Checker board titrations were done to optimize the concentration of each  antigen. The amount of recombinant EgAgB8/1, EgAgB8/2 and EPC1 antigens coated on each well of the 96-well microtitre plate was optimized to 1.0 µg /ml, 2.0 µg /ml and 2.0 µg/ml of coating buffer, respectively. The optimal dilutions of serum samples and anti-goat IgG-HRP conjugate (Sigma Chemicals, USA) used in the assay were 1:100-1:200 and 1:6000-1:12000, respectively for different antigens. Receiver Operating Characteristic (ROC) curve analysis was performed to determine the cut-off value for each recombinant antigen. Levels of sensitivity were plotted against the levels of one minus specificity at each cut-off point on a ROC curve. Cut-off values were selected that gave the highest sum of sensitivity (%) and specificity (%), as described by Amagai et al¹¹. The area under the ROC curve (AUC) was the parameter used to define the antigen’s discriminatory values between ELISA positive and negative animals.