Collection of worm and blood samples
A total of forty sheep (between 1-2 years old), with relatively high abomasal infections (harbored about 1000 to 2000 worms), were selected randomly from different flocks referred to the local industrial abattoirs in Shiraz (29.5926° N, 52.5836° E), Fars province, South of Iran. The abomasa cut and placed in warm 37 °C phosphate-buffered saline (PBS). Worms were then recovered, washed three times in PBS, pH 7.2, containing penicillin (10000 IU/ml) and streptomycin (10 mg/ml) and used for antigen extraction. A number of worms were morphologically identified according to previous descriptions for T. circumcincta . Regarding the prevalence of other trichostrongylid nematodes in the sampled area, efforts were made to ensure full recovery of worm burden to exclude abomasa with mixed infection. Prior to the slaughtering process, peripheral whole blood samples were collected from the jugular vein of animals. Sera were separated from the samples collected from the infected sheep and maintained at -20◦C until use. In addition, blood samples from inspected 10 non-infected animals (under 6 months of age) with no nematode infection were sampled as negative controls. Sample collections were performed after the consent to participate was obtained from farm owners.
Adult T. circumcincta worms were randomly selected from at least 2 farms and subjected to genomic extraction using a DNA extraction Kit (MBST, Iran). A primer set was used: F (5'-GCAGACGCTTAGAGTGGTGA-3') and R (5'-TCCTTGTTAGTTTCTTTTCCTCCG-3'), as described previously to identify the complete rDNA ITS-2 region in ostertagiine nematodes . The PCR reaction mix included 12.5 μl of PCR premix (Ampliqon, Denmark, Cat. No. A180301), 1 μl of each primer, 6.5 μl H2O and 4 μl of DNA as template. The cycling program consisted of an initial denaturation at 95°C for 5 min, 94°C for 30 sec, followed by 35 cycles of 60°C for 30 sec, the extension at 72°C for 30 sec and the final extension at 72°C for 10 min. The products were sequenced (ABI 3730 DNA analyzer; Bioneer, Korea) and compared with other available sequences in NCBI using BLAST search. The sequence data was aligned with homologous sequences existing in the GenBank using Clustal W program by M EGA 6 software . The phylogenetic tree was constructed by maximum likelihood (ML) method and analyses was carried out using the Kimura 2-parameter distance estimate .
Preparation of T. circumcincta somatic and ES antigens
Somatic antigens were extracted from washed adult specimens. A total of (about) 1000 worms were separately homogenized by tissue grinding, sonicated in 10 mL PBS pH 7.2 and centrifuged at 12000 rpm for 15 min at 4 ˚C. The extract was filtered through 0.45 mm filters and finally stored at –20 ˚C until use.
In order to prepare the ES products, freshly isolated adult worms (n~1000) were washed four times in normal saline and subsequently in PBS, pH 7.2, containing penicillin (1000 IU/ml) and streptomycin (1 mg/ml). Worms with high rates of motility were maintained in a sterile culture ﬂask at a density of approximately 100-200 worms/mL of the culture medium (RPMI-1640; Gibco, ThermoFisher Scientific, USA) with penicillin G potassium and streptomycin (1000 IU/ml and 1 mg/ml) and incubated for 24 hours in 5.0% CO2 at 37 ˚C. Supernatants were collected and centrifuged at 12000 rpm for 15 min at 4 ˚C. The supernatant was then dialysed against PBS over a period of 24 h, and concentrated using freeze-drying process (Lyophilisation) (Zirbus, Netherlands). Protein content was estimated by the Bradford method  and stored at –20 ˚C until use.
Preparation of rabbit polyclonal antisera
Ten adult male rabbits of a commercial New Zealand White strain with average weight of 2 ± 0.2 Kg were maintained under constant conditions with access to water and food in the animal house, School Veterinary Medicine, Shiraz University. Rabbits were adapted to new conditions for at least two weeks prior to immunization and divided in to 5 groups. Groups 1 to 4 received somatic and ES antigens of T. circumcincta, H. contortus, M.marshalli and P. rufescens and group 5 was defined as control. Blood samples (of about 5 ml) were collected from each rabbit before first injection (day zero) and used as negative controls. According to vaccination protocol (Fig. 1), 300 mg antigen, in a volume of 1 ml of PBS, emulsiﬁed with 1 ml of Freund’s complete adjuvant (Sigma, USA) was first injected subcutaneously at day zero. This followed by four boosters comprised of 150 mg antigen in a volume of 1 ml of PBS emulsiﬁed with 1 ml of Freund’s incomplete adjuvant (Sigma, USA). Boosters were given at one week intervals. The control group (group 5) was administered with 1 mL of sterile PBS plus 1 mL adjuvant. Rabbits were bled one week after final booster (at day 35), sera were collected and stored at –20 ˚C until use. The polyclonal hyper-immune serum was evaluated against somatic and ES antigens by indirect ELISA and Western blotting.
Somatic and ES antigens were separated by sodium-dodecyl-sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) based on Laemmeli's method . The samples were mixed with an equal volume of a sample buffer and boiled for 5 min. They then were added to each well of a 5% stacking gel and 12% separating gel. Electrophoresis was run at 100 V for 4 hr under reducing conditions using electrophoresis apparatus (Paya Pajoohesh Pars, Tehran, Iran). Stained molecular mass standards (Cinnagen PR911654 [SL7012]) ranging from 11 to 180 kDa were used. SDS-polyacrylamide gel was stained for protein visualization with 0.05% Coomassie brilliant blue (Sigma, USA.).
For immunoblotting, proteins were first electrophoresed on 12% SDS-polyacrylamide gel. Western blotting was carried out as described previously  with modifications. Proteins were transferred onto a nitrocellulose membrane for 1.5 hr. After blocking overnight at room temperature (RT) with 5% skimmed milk in PBS, nitrocellulose membrane stripes were cut, washed with PBST and incubated with serum sample at RT for 1 hr. Sera samples from the immunised rabbits and the infected sheep were diluted as 1:50 and 1:10, respectively. After washing, anti-rabbit and anti-sheep conjugate peroxidase (Sigma, USA) diluted in PBS-T (1:2000 - 1:1000) was added and incubated with shaking for 1 hr at RT. Finally, the membrane stripes was washed and placed into a substrate solution 0.05% diaminobenzidine in 50mM Tris pH 7.4 containing 0.05% H2O2. (DAB/ H2O2) (Sigma, USA).
The reactivity of sera from sheep and immunized rabbit against T. circumcincta somatic and ES antigens was tested against the same products prepared from prevalent field isolates of H. contortus, M. marshalli and a lungworm species, P. rufescens.
Enzyme Linked Immunosorbent Assay (ELISA)
The checkerboard titration for determination of different dilutions of antigen, sera, and conjugate was done . An indirect ELISA (iELISA) was carried out and optimized with serum samples. 96-well microplates were incubated with 100 μl/well of antigen at 1.1 mg/ml for somatic and 7.5 mg/ml for ES proteins in 50 mM carbonate bicarbonate buffer (pH 9.6) at 4ºC overnight. After washing three times with PBS containing 0.05% (v/v) Tween 20 (washing buffer), plates were blocked with 200 μl/well of the blocking buffer (PBS at pH 7.2 with 1% Bovine serum albumin) at RT for 2h. Follow 3 times washings, 100 μl of diluted sheep and rabbit sera 1:2 in 1% BSA were incubated at RT for 1 h. The plates were washed as described above and 100 μl/ well of horseradish peroxidase anti-sheep and anti-rabbit IgG conjugate (Sigma, USA) diluted at 1:5000 were added and incubated for 1h at RT. The plates were washed three times and 100 µl of the substrate buffer contains (0.02 g Ortho-Phenylenediamine) (Sigma, USA) in citrate buffer and 30% H2O2 were added to the plate wells. Finally, the optical density (OD) were obtained from an ELISA reader (Immunoskan BDSL, Thermo Lab. Systems, Finland) at 450 nm. All samples were run in duplicate. A pool containing sera of 10 naturally infected sheep with immune-reactivity against T. circumcincta (somatic and ES) antigens in western blot test was used as the positive control. Because no serum samples were available from parasite naïve sheep mono-infected with other GINs, the specificity of the method was checked by cross reactivity test using rabbit hyperimmune sera as described earlier.
In order to normalizing the OD estimates in the ELISA, values were quantified as the relative ODR according to the formula: ODR= (OD–N)/(Ps–N), where N and Ps are the mean absorbance values for negative and positive controls. The rates of sensitivity and specificity were evaluated as sensitivity = (true Ps)/(true Ps + false N) × 100 and specificity = (true N)/(true N + false Ps) × 100. In order to determine the best cut-off values, receiver operating characteristics (ROC) analysis was performed and the point showing maximum percents of the sensivity and specificity were considered. The SPSS software (Version 16.0) was used for the statistical analyses and the GraphPad Prism 8 for drawing graphs.