Mite collection and RNA extraction
P. ovis var. cuniculi were harvested from an infested New Zealand White rabbit maintained at the Department of Parasitology, Sichuan Agricultural University (Sichuan, China). About 300 mites, a pool of larvae, nymphs and adults, were collected and processed for the total RNA extraction using a MiniBest universal RNA extraction kit (TaKaRa, Dalian, China).
Cloning of two PsoSP genes
Total RNA was converted into cDNA following the protocols of the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). The PsoSP1 and PsoSP2 genes were amplified from cDNA by using the following primers: 5’-CGGGATCCGCTCATGTTGGTCAACATC-3’ (forward) and 5’ CCAAGCTTTTAAAAATCATGAATTTCACC-3’ (reverse) for PsoSP1 with underlined restriction enzymes of BamH I and Hind III, and 5’- CGGGATCCTGAATGCGAATTCATTGCTG-3’ (forward) and 5’-CCCTCGAG TCAAAATCCATGCATTTCACC-3’ (reverse) for PsoSP2 with underlined restriction enzymes of BamH I and Xho I. The DNA fragments were purified and ligated into the pMD19-T (TaKaRa, Dalian, China). Then, the plasmids were transformed into Escherichia coli strain DH5α (TIANGEN Biotech Co. Ltd., Beijing, China), and sequences were confirmed by Invitrogen Biotechnology Company (Shanghai, China).
Sequence analysis
DNAMAN version 7.0 was applied to compare the similarity between paralogous genes, and SignalP 5.0 (http://www.cbs.dtu.dk/Services/SignalP/) was used to predict signal peptides. Transmembrane regions were analyzed using the Transmembrane Prediction Server (http://www.cbs.dtu.dk/services/TMHMM-2.0). B-cell epitopes were predicted by BaCelLo (http://gpcr.biocomp.unibo.it/bacello/pred.htm). The serine protease inhibition domains were analyzed by Inter-ProScan EMBL-EBI Software (http://pfam.xfam.org/). Amino acid sequences were aligned using MEGA5.0. Secondary structure predictions were performed by JPred 4.0 (http://www.compbio.dundee.ac.uk/jpred/). The neighbor-joining (NJ) tree, including values of 1000 replications resampled tests, was constructed by MEGA 5·0 software [16].
Expression and purification of PsoSP
The correct plasmids of two PsoSP genes were digested and ligated into the pET32a (+) expression vector (Invitrogen, Beijing, China), respectively. Then, the resulting constructs were transfected into E. coli BL21 (DE3) and induced by 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) at 20 ℃ for 10 h. The recombinant PsoSP1 (rPsoSP1) and PsoSP2 (rPsoSP2) were harvested from the supernatant and inclusion body, respectively, and further purified by a Bio-scale TM Mini Nuvia TM IMAC Ni-Charged column (Bio-Rad, California, USA) using a step-wise elution with 50, 100, 200, 300 and 400 mM imidazole. The eluted fractions were concentrated by Amicon Ultra Centrifugal Filter Devices (Millipore, Billerica, MA, USA).
Rabbit sera
Fifty P. ovis var. cuniculi-positive rabbit sera were collected from a farm located in Chengdu, Sichuan, China. All rabbits were confirmed P. ovis var. cuniculi-positive by observation of ear scab and skin scrapings by microscopy (Ochs et al., 2001). Twenty-five negative sera from P. ovis var. cuniculi-free rabbits were obtained from a farm without a history of psoroptic mange. For cross-reaction testing, another 30 sera included Sarcoptes scabiei-positive sera, Eimeria spp.-positive sera, and Cysticercus pisiformis-positive sera (ten/group) were provided by the Department of Parasitology, Sichuan Agricultural University.
Preparation of polyclonal antibodies and Western blotting
Polyclonal antibodies of anti-rPsoSP1 and anti-rPsoSP2 from rabbits were obtained by experimentally immunized with purified rPsoSP1 and rPsoSP2, respectively. The rabbits were immunized with about 1 mg purified recombinant protein mixed with an equal volume of Freund’s complete adjuvant (Sigma-Aldrich) by subcutaneous injection at day 0. The next two immunizations were performed with mixture 1 mg protein and the same amounts of Freund’s incomplete adjuvant at days 7 and 14, respectively. At 7 days after the final immunization, sera were collected from rabbits via the marginal ear vein, and then purified by HiTrap Protein A affinity chromatography (Bio-scale TM Mini UNOsphere SUPrA TM Cartridge) to obtain the IgG of anti-rPsoSP1 and anti-rPsoSP2.
Two purified rPsoSP proteins were separated by 12% SDS-PAGE and transferred to the nitrocellulose membranes using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad). The membranes were blocked using 5% skim milk powder for 2 h. After three times of 5-min washes with TBST (0.02 M Tris-HCl, pH7.6, 0.15 M NaCl, 0.05% Tween-20), membranes were incubated with rabbit anti-P. ovis var. cuniculi antibody or anti-rPsoSP1 IgG or anti-rPsoSP2 IgG (1:150 v/v) overnight at 4 °C. Non-infested rabbit serum was used as a negative control. After washing three times with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:1000 dilution; Boster Bio-project Co. Dalian, China) for 1 h at room temperature. Following three washes with TBST, the signal was detected using an Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen, Beijing, China).
Immunolocalisation of PsoSP1 and PsoSP2 in adult female P. ovis var. cuniculi
Adult female mites were fixed in 0.8% (w/v) agarose, and then soaked in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 h to investigate the tissue localization of PsoSP1 and PsoSP2. The mites were processed according to the paraffin-embedded tissue technique and sliced into 5 µm histological section using a rotary microtome (Leica, Frankfurt, Germany). After dewaxing and rehydration, parts were treated with 0.01 M citrate buffer and incubated at 37 °C with 5% (w/v) skimmed milk for 4 h, then incubated with purified rabbit anti-rPsoSP1 IgG or anti-rPsoSP2 IgG or pre-immune IgG (1:200 v/v) overnight at 4 °C. After three times of 5-min washing with PBS, sections were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:200 v/v) at 37 °C for 1 h in the dark and finally visualized by a fluorescent microscope (BX53, Olympus, Japan).
Transcriptional profiles of PsoSP at different life stages of P. ovis var. cuniculi
To analyze stage-specific gene expression profiles of PsoSP1 and PsoSP2 in P. ovis var. cuniculi, the total RNA was extracted from larvae, nymph, and adult (male/female) mites using a MiniBest universal RNA extraction kit (TaKaRa), resptectively. Relative gene expression was evaluated by two-step qRT-PCR with the following primers: PsoSP1, 5’-TGGCAGCAGTGGATCAGAATCATC-3’ (forward) and 5’-AATGCAACAGCAACACTGTATGGC-3’ (reverse); PsoSP2, 5’-TCCTACATACACGTCCATCAACA-3’ (forward) and 5’-TGGTACAATAGCGACGGCTG-3’ (reverse). The β-actin gene was used as a housekeeping control to correct the relative fluorescence signal of the target genes using primers 5’-TGAATTGCCTGATGGTCAAG-3 (forward) and 5’-TGGCGAACAAGTCTTTACGG-3 (reverse). Gene transcription was assessed according to the manufacturers’ recommendations of the real-time PCR System (LightCycler® 96 System, Roche, Switzerland) and the SYBR Premix Ex Taq II Kit (TaKaRa, Dalin, China). The qPCR experiments were carried out in 25 µL reactions containing 12.5 µL SYBR premix Ex Taq II, 1 µL of each primer, 2µL cDNA and 8.5 µL ddH2O. Each sample was performed in triplicate. An equal volume of ddH2O replaced the template cDNA as blank control. Thermal cycling was performed as follows: 95 °C for 30 s, 95 °C for 5 s, 58 °C for 30 s, then followed by 40 cycles at 95 °C for 0 s, 59 °C for 15 s, and 95 °C for 0 s. Melting curves were plotted, and relative expression levels of the target genes were calculated by the 2− ΔΔCt method.
Establishment of an Indirect ELISA (iELISA)
The establishment of iELISA was performed as described by Crowther and Walker [17]. The concentrations of antigen and primary serum samples were determined by the checkerboard titration tests. Briefly, the purified proteins were diluted two-fold in 0.1 M carbonate buffer (pH 9.6) to the different concentrations and coated in 96-well plates with 100 µL/well overnight at 4 °C. The dishes were washed three times with PBS containing Tween-20 (PBST, pH 7.4) (5 min per wash), then incubated with 5% (w/v) skim milk powder at 37 ℃ for 90 min. After washing three times, 100 µL of two-fold gradient dilution of P. ovis var. cuniculi-positive and negative serum samples (diluted in PBS ranging from 1:20 to 1:320) were added and incubated at 37 ℃ for 1 h. The plates were washed 3 times with PBST and incubated 1 h at 37 ℃ with 100 µL HRP-labeled goat anti-rabbit IgG (1: 3000 dilution with 0.01 M PBS) (Boster Bio-project Co., Wuhan, China). After 4 times washing with PBST, 100 µL of TMB chromogenic solution (TIANGEN, Beijing, China) was added at 37 °C for 20 min, then the reaction was stopped with 100 µL / well of 2 M H2SO4. Optical densities (OD) were read at 450 nm by a microplate reader (Thermo Scientific, Pittsburgh, PA, USA). The optimal working conditions were determined with the highest P/N (positive/negative serum) value. The cut-off value of iELISA was determined as the mean OD450 value plus three standard deviations (SD) using 25 negative serum samples from naïve rabbits [1].
To further evaluate the feasibility of the iELISA, 50 P. oivs var. cuniculi-positive serum samples were assessed by the iELISA, and the sensitivity was calculated as (ELISA positive × 100) / true P. ovis var. cuniculi-positive [1]. Thirty serum samples from rabbit infected with S. scabiei, Eimeria spp and C. pisiformis (10 samples for each species) were used to evaluate the cross-activity of the iELISA. Twenty-five negative serum samples from naïve rabbits and 30 serum samples in the cross-activity assay were used to determine the specificity of the iELISA, which was calculated as (ELISA negative × 100) / real P. oivs var. cuniculi-negative [1]. After that, the area under the receiver operating characteristic curve (AUC), a graph of the sensitivity (true positive rate) versus 1-specificity (false positive rate), was calculated by MedCalc 19.0.7 [18].
The repeatability (intra-assay variability) and reproducibility (inter-assay variability) of the iELISA were evaluated using three P. ovis var. cuniculi-positive serum samples, substantially as previously described [19].
The experimental infestation of rabbits with P. ovis var. cuniculi and serological testing using the established iELISA
Rabbits infected with P. ovis var. cuniculi were performed strictly as previously described [3]. Briefly, ten 3-month-old naive New Zealand rabbits (5 females and 5 males) were infested on the aural region with approximately 200 mites of mixed stages of P. ovis var. cuniculi. Three non-infested rabbits were applied as controls. Serum samples from 13 rabbits were collected from the marginal ear vein at weeks 0, 1, 2, 3, and 4. Afterward, in a total of 65 serum samples (50 from the P. ovis var. cuniculi infestation rabbits and 15 from the non-infestation rabbits) were examined by the establishment optimal iELISA method. Each serum sample in triplicate was analyzed in one ELISA plate, and positive and negative controls were contained in the dishes.
Statistical analysis
All data are represented as mean ± standard deviation (SD), and statistical differences between groups were evaluated using Mann-Whitney U tests in SPSS software v.17.0. P-values < 0.05 were considered as statistically significant.