All experimental protocols were approved by the Science and Technology Agency of Jiangsu Province (Approval ID: SYXK (SU) 2010–0005).
Animals and Parasites
Local crossbred female goats (n = 35), aged approximately 6 months, were bought from a farm in Xuyu city of Jiangsu province and kept in animal house of Nanjing Agricultural University (NAU) under controlled conditions. All goats were orally administered Levamisole (8 mg/kg body weight) at two week intervals to remove natural parasitic infections. Microscopic analysis of fecal samples was performed twice per week for helminth eggs. Helminthes free goats were used in further experiments.
Goats were divided randomly into group 1 (n = 5), group 2 (n = 28) and group 3 (n = 2). Group 1 (experimental) was artificially orally infected with 5000 infective larva of H. contortus (L3) and group 2 with 8000 L3, while group 3 was kept un-infected as control. Fecal samples were examined to confirm H. contortus infection following the standard parasitological methods. After completion of the study, infected goats were euthanized by injecting sodium pentobarbital (>150 mg/kg) intravenously .
Female SD rats of 150 g body weight (n = 6) were bought from the Experimental Animal Center of Jiangsu, PR China (Certified: SCXK 2008–0004). Rats were randomly divided into two groups experimental (n = 3) and control group (n = 3) to collect polyclonal antibodies. Rats were kept in sterilized room and supplied with food and water.
Infected larvae of H. contortus (L3) were collected for experimental infection through conventional method . Briefly, feces from H. contortus infected-goat were collected, crushed, mixed with water and combined with vermiculite to make mixture moist. The pan was covered with aluminum foil having several holes to allow air flow and put at room temperature for ten days. To recover, larvae mixture was filtered through cheesecloth and identified larva microscopically. The larvae were preserved at 4°C in penicillin G added water until use.
Blood samples were collected from helminth free goats of all groups before challenging the infection to evaluate the cut-off value and specificity. Serum samples were collected from group 1 at specified days; one day prior challenging infection (control) and 7 to 103 days post challenging infection with week interval for antibody detection. Serum samples from group 3 were obtained after confirmation of H. contortus infection by examination of fecal samples and were used to evaluate the sensitivity. Sera were stored at –20°C after preparation. Various antisera against Trichinella spiralis (T. spiralis),, Fasciola hepatica (F. hepatica) and Toxoplasma gondii (T. gondii) (provided by Professor Yan) were also used to evaluate the specificity.
Purification of recombinant protein of H. contortus
The recombinant plasmid of CS (pET–32a+rHc-CS) was provided by MOE joint international Research Laboratory, Preventive Veterinary Medicine, NAU (GenBank: CDJ84294.1) and purified by standard protocol described previously . Briefly, transformation of recombinant plasmid into E.coli BL21 (DE3) was performed and cultured in ampicillin (100µg/ml) containing LB medium (Luria Bertini). After that, induction of protein expression was done at 37°C using IPTG (Isopropyl β-D-thiogalactopyranoside; Sigma-Aldrich) to make 0.6 (OD600). Culture was centrifuged at 4500rpm for 15 min and supernatant was discarded. Pallet was lysed by using lysozyme (10µg/ml Sigma-Aldrich) followed by sonication. 12% (w/v) SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was used to examine the sonication products. Manufacturer’s instructions were practiced to purify recombinant protein using Ni2+ nitrilotriacetic acid column (GE Healthcare, USA). The rHc-CS protein purity and quantification were performed by 12% SDS-PAGE with Coomassie blue stain and Bradford method respectively . Protein toxins were removed utilizing Toxin EraserTM Endotoxin Removal kit (GeneScript, USA).
Immunoblotting plays preliminary role in selection of target protein to diagnose the infection of H. contortus and to evaluate the immunogenicity and immunoreactivity of antigen . Complete Freund’s adjuvant mixed with rHc-CS protein (300μg) was injected subcutaneously in SD rats of experimental group to obtain polyclonal antibodies. After 2 weeks, three more doses of this protein mixed with Freund’s incomplete adjuvant were injected at 1-week interval. After one week of the last dose, SD rats were anesthetized with 25% isoflurane (inhaling anesthesia) by open drop method  and blood samples were collected from eye. Finally, SD rats were euthanized by head dislocation. The serum samples from experimental group and control group were prepared and used in immunoblotting to evaluate the immunoreactivity of rHc-CS. The sera from normal rats were also used as negative control.
Immunoblotting analysis was also performed to evaluate the antigenic characteristics of recombinant H. contortus CS (rHc-CS) at early stage as well as late stage of H. contortus infection in sera of experimentally infected goats (Group 1). The purified rHc-CS was first separated on 12% SDS-PAGE and transferred onto PVDF (polyvinyl difluoride membrane, Millipore, Billerica, USA) with the help of semi dry system (Novablot Hoefer, USA) and transfer solution (Tris 48 mM, glycine 39 mM, SDS 0.0375%, methanol 20%). The membrane was blocked with 5% skimmed milk diluted in TBS-T (Tris-Buffered Saline containing 0.05% Tween 20) for 2h at 37℃. The PVDF membrane was cut into five strips, washed (3 times) and incubated with anti-H. cortortus 1:100 diluted goat serum (primary antibody) for 2h at 37℃. Following 3 washes with TBS-T the strips were incubated with 1:4,000 diluted Horseradish Peroxidase (HRP) conjugated rabbit anti-goat IgG (Sigma, Hilden, Germany). Subsequently, the strips were washed five times and immunoreactions were observed by virtue of substrate (Tanon™ High-sig ECL Western Blotting). Upon each blood sampling day, the whole immunoblotting practice was repeated whereas the primary antibody differed. All anti -H. contortus sera separated from infected and non-infected goats (Group 1), on those sampling days were taken as the primary antibody. Immunoblotting also helped to check the specificity of rHc-CS against infected sera of T. spiralis, F. hepatica and T. gondii as primary antibody.
Optimization of indirect-ELISA
Indirect-ELISA was performed to assess the immunodiagnostic potential of rHc-CS . The rHc‑CS indirect-ELISA was optimized by evaluation of numerous format variables including working dilution of rHc-CS antigen (4.5 to 0.07 μg/well), serum samples (1:25 to 1:200 dilutions), secondary antibody HRP conjugated rabbit anti-goat IgG and working time of antigen coating, blocking, serum incubation and tetramethylbenzidine (TMB) reaction (Table 1). Furthermore, best blocking buffer was also determined using same method. The optical density (OD) values derived from the checkerboard were calculated and showed as the positive to negative (P/N) ratio . The highest P/N (OD450 of infected serum / OD450 of non-infected serum) value (>2.1) was used to optimize the experimental conditions.
Establishment of Indirect ELISA
In order to evaluate the diagnostic potential and to ratify the immunoblotting results of the rHc-CS, an indirect-ELISA with optimal conditions was performed as discussed previously . Briefly, the reaction was performed in 96-well flat bottom without lid high binding plates (Costar, Bodenheim, USA) in triplicate. The wells were coated with 100μl of diluted rHc-CS in 0.05M carbonate buffer solution (CBS; pH 9.6) followed by overnight incubation at 4°C. Next day after washing, all wells were blocked with 100μl BSA (bovine serum albumin) for 2h at 37℃. Then washed three times with TBS-T, 100μl of positive and negative goat sera, diluted in blocking buffer, were added to known well and incubated at 37 °C. After 2h incubation, wells were washed again (3 times) and incubated with 100μl of diluted secondary antibody HRP conjugated rabbit anti-goat IgG at 37℃ for 1 h. Then the last three washings were performed to add 100μl TMB in each well and incubated for 10 min in the dark at room temperature. The color reaction was stopped by adding 100μl/well of 2M H2SO4. The OD of wells was read at a wavelength of 450 nm using micro plate reader (Thermo Fischer Scientific, Waltham, MA, USA). Moreover, to investigate the further feasibility of indirect ELISA, serum samples (n = 33) from goats infected with H. contortus were used to calculate diagnostic sensitivity and serum samples (n = 35) from non-infected goats were evaluated to calculate diagnostic specificity. The sensitivity and specificity were defined using following formula described previously .
[Due to technical limitations, this equation is only available as a download in the supplemental files section.]
Additionally, a total of 12 serum samples (4 samples for each parasite) collected from goats infected with T. spiralis, F. hepatica and T. gondii were used to evaluate the cross-reactivity. This experiment was performed in duplicate.
Determination of cut-off value
Total 35 negative blood sera were used to determine the cut-off value. The cut-off value was calculated by taking “mean absorbance values of known negative sera + (3× standard deviation)" . The experiment was repeated twice and for the interpretation, any goat sera that had an OD450 value greater than the cut off value was considered as seropositive while OD450 value lower than cut-off value was considered as sero-negative .
The Receiver operating characteristic (ROC) analysis was used to simulate the influence of different cut-off values on the sensitivity and specificity of the test . ROC curves were obtained using statistical software MedCalc (version 15; http://www.medcalc.be). Statistical analysis of the data was evaluated by using the Graph Pad Prism™ v6 07 software.