Molecular characterization and serodiagnostic potential of two serpin proteins in Psoroptes ovis var. cuniculi

Background Psoroptes ovis var. cuniculi is a common ectoparasite of wild and domestic rabbits worldwide that causes economically devastating losses in commercial rabbit husbandry and significantly affects the overall health of rabbits. Serine proteinase inhibitor (serpin) is present in almost all organisms that are involved in host–pathogen interactions, inflammatory responses, and reproductive development, among others. However, very little research has been carried out on P. ovis var. cuniculi serpins. Methods Two serpin genes of P. ovis var. cuniculi (Pso c 27 and PsoSP2 cDNAs) were cloned and molecularly characterized. The transcriptional profiles and tissue localization of these two serpins in P. ovis var. cuniculi were investigated by quantitative real-time PCR and immunohistochemistry, respectively. The potential function of recombinant Pso c 27 and PsoSP2 (rPso c 27 and rPsoSP2) in the serodiagnosis of P. ovis var. cuniculi infestation in rabbits was evaluated using a newly devleoped indirect enzyme-linked immunosorbent assay. Results Both the 523-residue Pso c 27 and the 240-residue PsoSP2 proteins contained typical serpin domains and signatures. Both Pso c 27and PsoSP2 cDNAs were expressed throughout the life-cycle; specifically, the cDNAs showed significantly higher expression in female mites than in larva, nymph, and male mites (Pso c 27: F(3, 8) = 1935.953, P < 0.0001; PsoSP2: F(3, 8) = 660.669, P < 0.0001). The native Pso c 27 and PsoSP2 proteins localized in the ovary and mouthparts of adult female mites, respectively. Compared to rPsoSP2, rPso c 27 showed better diagnostic efficiency, with higher values of sensitivity, specificity, and area under the receiver operating characteristic curve (rPso c 27 vs rPsoSP2: 96.0 vs 90.0%; 90.91 vs 78.18%; 0.988 vs 0.964, respectively). Moreover, rPso c 27 showed seropositivity in 80% of the rabbits as early as the 2 weeks post-infestation, prior to visible clinical signs and microscopy-positive of skin scrapings. Conclusions These results suggest that these two serpins may play essential roles in reproductive development, serum-feeding, and pathogenicity of P. ovis var. cuniculi. Compared to PsoSP2, Pso c 27 appears to be a potential antigen for serodiagnosis of P. ovis var. cuniculi infestation in rabbits, especially at the early stage of infestation.


Background
Psoroptes ovis var. cuniculi is a common ectoparasite of wild and domestic rabbits worldwide [1,2]. This mite causes psoroptic mange in rabbits, which mainly presents as intense cutaneous inflammation, extreme pruritus, and crusted skin lesions [1,2]. It signficantly affects the health welfare of rabbits and causes severe economic losses in commercial rabbit husbandry [2,3].
The obligate ectoparasite P. ovis var. cuniculi is a non-burrowing mite that spends its entire life on the surface of host skin [4] where it feeds on serous fluids, lymph, and red blood cells [5]. The mite produces essential proteins to resist the host complement system to ensure successful feeding and self-proliferation. It also excretes allergens to promote the subsequent cutaneous inflammatory response [6,7]. Serine protease inhibitor (serpin) is expressed in almost all organisms. In arthropods, it has shown to possess a variety of fundamental physiological functions including anticoagulation, regulation of the inflammation response, and reproductive development, among others [8]. It also plays an essential role in the host-pathogen interaction [9]. Additionally, serpin may serve as a promising diagnostic antigen or vaccine candidate [10,11].
Recently, our analysis of transcriptomic data revealed that serpins exist in P. ovis var. cuniculi [12], but to our knowledge no further research on P. ovis var. cuniculi serpins has been reported to date. Analysis of these transcriptomic data led to the identication of two serpin genes of P. ovis var. cuniculi (Pso c 27 and PsoSP2 cDNAs) [12]. In the present study we focused on the function of these genes. We cloned and expressed the two recombinant Pso c 27 and PsoSP2 in prokaryotic expression vectors and performed sequence analysis. We also investigated the transcriptional profiles as well as tissue localization in mites, and their potential efficiencies in the diagnosis of P. ovis var. cuniculi infestation in rabbits were determined by indirect enzyme-linked immunosorbent assay (iELISA). This is a preliminary study aimed at elucidating the roles of these two proteins in P. ovis var. cuniculi and increasing our understanding of their functions. pooled larvae, nymphs, and adults, were collected and processed for total RNA extraction.

Expression and purification of two recombinant serpin proteins
Total RNA was converted into cDNA using the Prime-Script RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China). The two serpin genes were amplified from cDNA using the following primers: 5′-CGG GAT CCG CTC ATG TTG GTC AAC ATC-3′ (forward) and 5′-CCA AGC TTT TAA AAA TCA TGA ATT TCA CC-3′ (reverse) for Pso c 27, where the underlining indicates restriction enzymes BamHI and HindIII; and 5′-CGG GAT CCT GAA TGC GAA TTC ATT GCT G-3′ (forward) and 5′-CCC TCG AGT CAA AAT CCA TGC ATT TCA CC-3′ (reverse) for PsoSP2, where the underlining indicates the restriction enzymes BamHI and XhoI. The cDNA fragments were sub-cloned into the pET32a(+) plasmid (Invitrogen, Beijing, China). The recombinant proteins were expressed in Escherichia coli BL21 (DE3) and purified as described previously by Gu et al. [2]. The eluted fractions were concentrated using an Amicon Ultra Centrifugal Filter unit (Millipore, Billerica, MA, USA). Two purified serpin proteins were detected by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Rabbit sera
Fifty P. ovis var. cuniculi-positive rabbit sera were collected from a farm located in Chengdu, Sichuan, China. Positivity for P. ovis var. cuniculi was confirmed in all rabbits based on the observation of ear scabs and micropscopic study of skin scrapings [14]. 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, an additional 30 sera samples, including Sarcoptes scabiei-positive sera, Eimeria spp.positive sera, and Cysticercus pisiformis-positive sera (ten samples of each) were provided by the Department of Parasitology, Sichuan Agricultural University.

Preparation of polyclonal antibodies and western blotting
Polyclonal antibodies were obtained by experimental immunization with purified rPso c 27 and rPsoSP2, respectively. The products were raised following the procedures of Gu et al. [2], with slight modification. Briefly, rabbits were immunized with about 1 mg purified recombinant protein four times by subcutaneous injection. Sera were collected from the marginal ear vein before immunization and 7 days after the fourth infection, and then purified by HiTrap Protein A affinity chromatography (Bio-scale ™ Mini UNOsphere SUPrA TM cartridge; Bio-Rad, Hercules CA, USA) to obtain the immunoglobulin G (IgG) of anti-rPso c 27 and anti-rPsoSP2.
Two purified recombinant 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 then blocked using 5% skimmed milk powder for 2 h, following which they were washed three times (each wash 5 min) in TBST (0.02 M Tris-HCl, pH 7.6, 0.15 M NaCl, 0.05% Tween-20) and then incubated with rabbit anti-P. ovis var. cuniculi antibody, anti-rPso c 27 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 another three washes with TBST, the signal was detected using an Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen, Beijing, China).

Immunolocalization of two serpin proteins in adult female P. ovis var. cuniculi
The immunolocalization of two serpin proteins were performed as previously described [15]. Briefly, adult female mites were collected, embedded in paraffin blocks, and sliced into 5-μm histological sections. The sections were then treated with 0.01 M citrate buffer and incubated with purified rabbit anti-rPso c 27 IgG, anti-rPsoSP2 IgG, or pre-immune IgG (1:200 v/v). After three washes with phosphate buffered saline (PBS), sections were incubated with fluorescein isothiocyanate-conjugated goat antirabbit IgG (1:200 v/v) and visualized using a fluorescent microscope (BX53; Olympus Corp., Tokyo, Japan).

Transcriptional profiles of serpin at different life stages of P. ovis var. cuniculi
Total RNA was extracted from larvae, nymph, and adult (male/female) mites, respectively, using a MiniBest Universal RNA Extraction kit (Takara Bio Inc., Shiga, Japan). Relative gene expression was evaluated by a two-step quantitative real-time PCR (qRT-PCR) using primers (i) Pso c 27: 5′-TGG CAG CAG TGG ATC AGA ATC ATC-3′ (forward) and 5′-AAT GCA ACA GCA ACA CTG TAT GGC-3′ (reverse); PsoSP2: 5′-TCC TAC ATA CAC GTC CAT CAA CA-3′ (forward) and 5′-TGG TAC AAT AGC GAC GGC TG-3′ (reverse). The β-actin gene was used as a housekeeping control to correct for the relative fluorescence signal of the target genes, using primers 5′-TGA ATT GCC TGA TGG TCA AG-3′ (forward) and 5′-TGG CGA ACA AGT CTT TAC GG-3′ (reverse). Gene transcription was assessed according to the manufacturers' recommendations of the real-time PCR system (LightCycler ® 96 System; Roche Group, Basel, Switzerland) and the SYBR Premix Ex Taq II kit (TaKaRa). Each sample was performed in triplicate. An equal volume of ddH 2 O replaced the template cDNA as a blank control. Thermal cycling was performed as follows: one cycle at 95 °C for 30 s, 95 °C for 5 s, 58 °C for 30 s; followed by 40 cycles at 95 °C for 5 s, 59 °C for 15 s, and 95 °C for 1 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 [16]. The concentrations of antigen and primary serum samples were determined by the checkerboard titration tests. Briefly, the purified proteins were diluted twofold in 0.1 M carbonate buffer (pH 9.6) to the different concentrations and placed in 96-well plates at 100 μl/well overnight at 4 °C. The plates were washed three times in PBS containing Tween-20 (PBST; pH 7.4) (5 min per wash), then incubated first with 5% (w/v) skimmed milk powder at 37 °C for 90 min, then with 100 μl of the two-fold gradient dilution of P. ovis var. cuniculi-positive and -negative serum samples (dilution ranging from 1:20 to 1:320) at 37 °C for 1 h. The plates were subsequently washed 3 times and incubated 1 h at 37 °C with 100 μl HRP-labeled goat anti-rabbit IgG (1:3000 dilution with 0.01 M PBS) (Boster Bio-Project Co.). After 4 washes, 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 H 2 SO 4 . Optical densities (OD) were read at 450 nm by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The optimal working conditions were determined based on the highest positive : negative serum value. The cut-off value of iELISA was determined as the mean OD 450 value plus three standard deviations (SD) using 25 negative serum samples from naïve rabbits [2].
To further evaluate the feasibility of the iELISA, 50 P. ovis var. cuniculi-positive serum samples were assessed by the iELISA, and the sensitivity was calculated as (ELISA-positive samples × 100)/true P. ovis var. cuniculipositive serum samples [2]. Thirty serum samples from rabbits infected with S. scabiei, Eimeria spp., and C. pisiformis, respectively (10 samples for each species) were used to evaluate the cross-activity of the iELISA. Twentyfive P. ovis var. cuniculi-negative serum samples from naïve rabbits and 30 serum samples in the cross-activity assay were used to determine the specificity of the iEL-ISA, which was calculated as (ELISA-negative samples × 100)/true P. ovis var. cuniculi-negative serum samples [2]. The area under the receiver operating characteristic curve (AUC), a graph of the sensitivity (true positive rate) versus 1-specificity (false positive rate), was the calculated using MedCalc 19.0.7 [17].
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 [18].
The experimental infestation of rabbits with P. ovis var. cuniculi and serological testing using the newly developed iELISA Rabbits infected with P. ovis var. cuniculi were treated strictly as previously described [2]. Briefly, ten 3-monthold naive New Zealand rabbits (5 females, 5 males) were infested with P. ovis var. cuniculi, and three non-infested rabbits were used as controls. Serum samples from 13 rabbits were collected at weeks 0, 1, 2, 3, and 4. Afterwards, a total of 65 serum samples (50 from the P. ovis var. cuniculi-infestated rabbits and 15 from the noninfested rabbits) were examined by the newly developed optimal iELISA method. Each serum sample was tested in triplicate and analyzed in one ELISA plate, with positive and negative controls also contained in the plate.

Statistical analysis
All data are presented as the mean ± SD. And statistical differences between groups were evaluated using Mann-Whitney U-tests in SPSS software v.17.0. (IBM Corp. Armonk, NY, USA). P values of < 0.05 were considered to be statistically significant.

Sequence analyses of the two serpins
The 1572-bp open reading frame (ORF) in Pso c 27 cDNA (GenBank: MT707535) encodes 523 amino acids (aa), and the 723-bp ORF in PsoSP2 cDNA (GenBank: MT707536) encodes 240 aa. The Pso c 27 protein contains a signal peptide but no transmembrane region, whereas PsoSP2 appears to contain a transmembrane region but no signal peptide.
Pso c 27 and PsoSP2 shared 31.33-50.85 and 28.99-69.92% aa sequence identity with orthologs in other mites (Fig. 1). Interestingly, Pso c 27 and PsoSP2 shared 100% aa sequence identity with the reported serpin-like proteins of P. ovis PSOVI22g04610 and PSOVI22g04560, respectively [19] (Fig. 1). Pso c 27 consisted of 12 helices Psoroptes ovis (PSOVI22g04610) and P. ovis (PSOVI22g04560) were obtained from the Online Resource for Community Annotation of Eukaryotes (OrcAE) (https ://bioin forma tics.psb.ugent .be/orcae /overv iew/Psovi ). Helices are indicated with red horizontal bars. Horizonatal dark-green arrows along the sequence indicate sheets. Elements of secondary structure are labeled as: hA, hB, etc. A-helix, B-helix, etc., s1A, s2A, etc. strand 1 of the A β-sheet, strand 2 of the A β-sheet, etc. Consistent residues are highlighted with a dark-blue background, and consistent partial residues are highlighted with a light-blue background. B-cell epitopes are marked with a black box and three sheets, while PsoSP2 consisted of five helices and three sheets (Fig. 1a). A serpin domain was identified in the aa sequence from Arg78 to His493 for Pso c 27 and from Asn4 to Met237 for PsoSP2 [20]. Moreover, both proteins appeared to possess the specific serpin signature at the deduced aa sequences from 496 to 506 (LRFDHPFLYFV) for Pso c 27 and from 213 to 223 (LSFDHPFLYFL) for PsoSP2, respectively (Fig. 1a, b). The NJ tree revealed that Pso c 27 had the closest relationship with P. ovis-leukocyte elastase inhibitor-like protein

Expression and identification of two recombinant serpins
rPso c 27 were mainly present in the supernatant, with an expected size of ~ 75 kDa, whereas rPsoSP2 principally present in insoluble inclusion bodies, with an expected size of ~ 46 kDa (including ~ 18 kDa His-tag fusion peptide from pET-32a) (Fig. 3). Western blotting showed that rPso c 27 and rPsoSP2 reacted with both P. ovis var. cuniculi-positive sera and the correspondent anti-serum IgG from rabbits, but not with negative sera, revealing a favourable reactivity and antigenicity (Fig. 3).

Tissue localization of two serpins in adult female P. ovis var. cuniculi
Native Pso c 27 and PsoSP2 were located in the ovary and mouthparts of female mites, respectively (Fig. 4b, c). No fluorescence signal was observed in adult female mites using pre-immunized rabbit IgG antibodies (Fig. 4a).

Serodiagnosis potential of two recombinant serpin proteins
Using checkerboard titration, we determined that the optimal working conditions of the iELISA were 46.0 μg/ ml of rPso c 27, 64.5 μg/ml of rPsoSP2 for coated antigens, and a 1:100 dilution for rabbit sera. The cut-off values of OD 450 were 0.633 for rPso c 27 and 0.490 for rPsoSP2.

Serodiagnostic test of rabbits experimentally infested with P. ovis var. cuniculi
At 4 weeks post-infestation (p.i.), all infested rabbits were observed to have ear scabs. Skin scrapings also tested positive for P. ovis var. cuniculi. By rPso c 27-iELISA, the mean value of the anti-rPso c 27 level from the infestation group revealed an increase from 1 to 4 weeks p.i. (Fig. 8).
In the infestion group, anti-rPso c 27 positivity above the cut-off value was first detected in two of ten serum samples at 1 week p.i., following which the positivity gradually increased to 80% of samples (8/10) at 2 and 3 weeks p.i., then up to 100% of samples (10/10) at 4 weeks p.i. (Fig. 8). In the non-infestation group, the rate of anti-rPso c 27 antibody positivity remained below the cut-off value throughout the experiments.

Discussion
In the present study, two P. ovis var. cuniculi serpins were characterized, and the potential of their recombinant proteins was evaluated for serodiagnosis of P. ovis var. cuniculi infestation in rabbits. The Psoroptes mites used in the present study and our previous transcriptomic study are the same species, and the present findings confirmed the transcriptomic results of the earlier study in showing that two cloned serpin ORFs had 100% identity with the assembled serpin sequences in the transcriptome data [12]. The predicted aa sequence showed the low overall identity of serpins compared those in other mites; however, these two target proteins were identified as typical serpins due to the presence of such features as the serpin domain and serpin signature in the C-terminal end [20]. Pso c 27 shared 50.85% aa sequence identity with the newly characterized D. farinae Der f 27 allergen, which has been proven to orchestrate the pulmonary inflammatory response and mediate a Th2-type response in mice [21]. In addition, NJ analysis revealed that Pso c 27 is closely related to Der f 27. Based on the homology results and the genetic relationship between Pso c 27 and Der f 27, Pso c 27 may be considered to be an allergen of P. ovis var. cuniculi that is possibly associated with the instigation of the host cutaneous pro-inflammatory response [22]. This cutaneous inflammation has been shown to result in serum extravasation, thereby providing sufficient food for mite population growth, and to aggravate scabby lesions [5,14]. The expression of Pso c 27 and PsoSP2 in all stages of mites indicated that Pso c 27 and PsoSP2 possibly play an essential role in the development of P. ovis var. cuniculi. However, significant differences were seen for the transcription of Pso c 27 in the different life-cycle stages, and female mites showed the highest level of expression, with 347-fold change. In addition, the native protein was located in the ovary of female mites, indicating that Pso c 27 is possibly an essential factor in vitellogenesis [19,23]. This role of serpin in vitellogenesis has been proven in a recent study, which indicated that RNA interference (RNAi) of the serpin gene resulted in a reduction of yolk granule accumulation in Rhipicephalus haemaphysaloides [24].
Psoroptes mites are serum-feeding ectoparasites [5] and possess the ability to counter the host's complement attack. In this study, PsoSP2 showed homology to the S. scabiei serpin family genes (20.98-54.13% aa sequence identity), some of which have been confirmed to inhibit the activation of complement pathways [25,26]. Moreover, the native PsoSP2 localized in the mouthparts of female mites and its cDNA expression throughout the life stages of mites suggest that PsoSP2 may appear to be vital in mites for anti-complement activity to successful serum-feeding [5,9], and PsoSP2 could be a potential vaccine candidate.
Psoroptic mange spreads rapidly under crowded conditions and causes major morbidity in commercial rabbit husbandry in China [27]. Thus, timely diagnosis and treatment of P. ovis var. cuniculi infestation in rabbits are of paramount importance to reduce the risk of disease transmission and improve profitability. In China, the current diagnostic method for this disease, by examination of skin scrapings under the microscope, is extremely time-consuming and inefficient, especially for low densities of mites and sub-clinical infestations in rabbits. Thus, it is imperative to develop effective immunoreactive antigens for the rapid and accurate diagnosis of P. ovis var. cuniculi infestation in rabbits. Rabbits infested with P. ovis var. cuniculi can evoke a sero-specific antibody [1,28], and this sero-specific antibody has been induced at the early phase of parasite infestation when rabbits appeared asymptomatic [1,2]. Thus, the ELISA can be considered as an accurate method for detecting carriers of low densities of mites and/or sub-clinical infestations when compared with the microscopy of skin scrapings. In a previous study, serpin of Schistosoma mansoni was considered to be a promising species-specific diagnostic antigen in human schistosomiasis [10]. Therefore, in this study, we evaluated the serodiagnostic potential of rPso c 27 and rPsoSP2 by developing an iELISA. Compared to the rPsoSP2-iELISA, the rPso c 27-iELISA displayed better diagnostic efficiency, with higher values for sensitivity, specificity, and AUC (rPso c 27-rPsoSP2: 96.0-90.0, 90.91-78.18, and 0.988-0.964, respectively). Although rPso c 27 did show cross-reaction with sera from three of ten serum samples with S. scabiei infestations, the crossreaction between these two ectoparasites has been commonly shown in other studies [2,29]. Fortunately, these two mite species were effectively treated with the same acaricide [3,30]. Also, one in ten rabbits infested with S. scabiei, C. pisiformis, or Eimeria spp. showed a seroreaction with rPso c 27; however, their OD values were close to the cut-off value and appeared to markedly lower than those rabbits infested with P. ovis var. cuniculi (F (1, Fig. 7 The receiver operating characteristic (ROC) curves of the rPso c 27-iELISA and rPsoSP2-iELISA for the detection of antibodies against P. ovis var. cuniculi. The ordinate represents the sensitivity of the iELISA. The abscissa represents the 1-specificity of the iELISA. The purple line shows the mean area under the curve (AUC) plot of rPso c 27-iELISA, and the green line shows the mean AUC plot of rPsoSP2-iELISA 78) = 115.444, P < 0.0001). Moreover, the rPso c 27-iELISA was able to detect seropositivity in 80% (8/10) of rabbits as early as week 2 p.i., prior to visible clinical signs and microscopy-positive skin scrapings. In terms of its high sensitivity and specificity, Pso c 27 can be considered to be the more suitable candidate antigen for serodiagnosis of P. ovis var. cuniculi infestation in rabbits, especially at the early stage of infestation.

Conclusions
In conclusion, Pso c 27 and PsoSP2 cDNAs displayed the typical characterization of the serpin superfamily, possessing the regular serpin domain and signature. The gene expression of Pso c 27 and PsoSP2 were found during all life stages of mites, with a significantly high expression in adult female mites. Compared to rPsoSP2, rPso c 27 seemed to display a better diagnostic efficiency than PsoSP2 by iELISA, suggesting that Pso c 27 could be developed as a potential antigen for serological diagnosis of P. ovis var. cuniculi infestation in rabbits, especially at the early stage of infestation.