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

Background: Psoroptes cuniculi is a global common ectoparasite of wild and domestic rabbits and causes an economically devastating loss and serious welfare issues of commercial rabbit husbandry. Serine proteinase inhibitor (serpin) is present in almost all organisms that are involved in host-pathogen interactions, inammatory responses, and reproductive development, etc. However, the research on P. cuniculi serpins is still limited. Methods: In this study, two serpin genes of P. cuniculi (Pso c 27 and PsoSP2 cDNAs) were cloned, and the molecular characterization was analyzed. The transcriptional proles and tissue localization of these two serpins in P. cuniculi were investigated by quantitative real-time PCR and immunohistochemistry, respectively. The potential function of recombinant Pso c 27 and PsoSP2 (rPso c 27and rPsoSP2) in the serodiagnosis of P. cuniculi infestation in rabbits were evaluated by indirect enzyme-linked immunosorbent assay (iELISA). Results: Both of the 523 residue Pso c 27 and the 240 residue PsoSP2 proteins contained typical serpin domains and signatures. Both Pso c 27and PsoSP2 cDNAs expressed throughout the life-cycle, more specically, signicantly higher expression in female mites than the 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 localized in ovary and mouthpart of adult female mites, respectively. Compared to rPsoSP2, the rPso c 27 displayed better diagnostic eciency with higher values of sensitivity, specicity and the area under the receiver operating characteristic curve (AUC) (rPso c 27 vs rPsoSP2: 96.0 vs 90.0%; 90.91 vs 78.18%; 0.988 vs 0.964, respectively). Moreover, the rPso c 27 showed seropositive in 80% rabbits as early as the 2 weeks post-infestation (p.i.), prior to visible clinical signs and microscopy-positive of skin scrapings. Conclusions: These results suggested that these two serpins may play essential roles in reproductive development, serum-feeding, and pathogenicity of P. cuniculi. Compared to PsoSP2, Pso c 27 appeared as a potential antigen for serodiagnosis of P. cuniculi infestation in rabbits, especially at the early stage of infestation.


Background
Psoroptes cuniculi is a common ectoparasite of wild and domestic rabbits worldwide [1,2]. This mite causes psoroptic mange of rabbits, mainly characterized as intense cutaneous in ammation, extreme pruritus and crusted skin lesions [1,2]. Additionally, it causes severe economic losses and welfare issues in rabbits feeds on serous uids, lymph, and red blood cells [5]. Consequently, mite produces essential proteins to resist the host complement system for successful feeding and self-proliferation. Meanwhile, it excretes allergens to promote the subsequent cutaneous in ammatory response [6,7]. expressed in almost all organisms, has shown a variety of fundamental physiological functions in arthropods including anticoagulation, regulation in ammation response and reproductive development etc. [8]. It also plays an essential role in host-pathogen interaction [9]. Additionally, serpin may serve as a promising diagnostic antigen or vaccine candidate [10,11].
Recently, transcriptome analyses revealed that serpins existed in P. cuniculi [12], but beyond that, no research has been reported on P. cuniculi serpins. Therefore, we are highly interested in the function of two serpin genes of P. cuniculi (Pso c 27 and PsoSP2 cDNAs) which were identi ed based on our transcriptomic data [12]. In this study, we cloned and expressed the two recombinant Pso c 27 and PsoSP2 in prokaryotic expression vectors, performed the sequence analysis. Additionally, we also investigated the transcriptional pro les as well as tissue localization in mites, and their potential e ciencies in the diagnosis of P. cuniculi infestation in rabbits were accessed by indirect enzyme-linked immunosorbent assay (iELISA). This is a preliminary study with relevance to the roles of these two proteins in P. cuniculi, which lay the foundation for further understanding of their functions.

Mite collection and RNA extraction
Psoroptes 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.
Expression and Puri cation of two recombinant serpin proteins Total RNA was converted into cDNA using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China). The two serpin genes were ampli ed 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 with underlined restriction enzymes of 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 with underlined restriction enzymes of BamHI and XhoI. The cDNA fragments were sub-cloned into pET32a (+) (Invitrogen, Beijing, China). The recombinant proteins were expressed in Escherichia coli BL21 (DE3) and puri ed as the previous method described by Gu et al. [2]. The eluted fractions were concentrated by Amicon ultra centrifugal lter devices (Millipore, Billerica, MA, USA). Two puri ed serpin proteins were detected by 12% SDS-PAGE.
Rabbit sera Fifty P. cuniculi-positive rabbit sera were collected from a farm located in Chengdu, Sichuan, China. All rabbits were con rmed P. cuniculi-positive by observation of ear scab and skin scrapings by microscopy [14]. Twenty-ve negative sera from P. 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 were obtained by experimental immunization with puri ed rPso c 27 and rPsoSP2, respectively. The products were raised following slightly modi ed procedures described by Gu et al. [2]. Brie y, rabbits were immunized with about 1 mg puri ed recombinant protein four times by subcutaneous injection. Sera were collected via the marginal ear vein before immunization and 7 days after the fourth infection, and then puri ed by HiTrap protein A a nity chromatography (Bio-scale TM Mini UNOsphere SUPrA TM cartridge; BioRad, Hercules CA, USA) to obtain the IgG of anti-rPso c 27 and anti-rPsoSP2.
Two puri ed recombinant proteins were separated by 12% SDS-PAGE and transferred to the nitrocellulose membranes using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, Hercules CA, USA). The membranes were blocked using 5% skimmed milk powder for 2 h. After three 5 min washes with TBST (0.02 M Tris-HCl, pH7.6, 0.15 M NaCl and 0.05% Tween-20), membranes were incubated with rabbit anti-P. cuniculi antibody or 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 Bioproject 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 two serpin proteins in adult female P. cuniculi The immunolocalisation of two serpin proteins were performed as previously described [15]. Brie y, adult female mites were collected and sliced into 5 μm histological section, then were treated with 0.01 M citrate buffer and incubated with puri ed rabbit anti-rPso c 27 IgG or anti-rPsoSP2 IgG or pre-immune IgG The total RNA was extracted from larvae, nymph, and adult (male/female) mites using a MiniBest universal RNA extraction kit (TaKaRa), respectively. Relative gene expression was evaluated by a two-step qRT-PCR with the following primers: 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 the relative uorescence 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, 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: 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 -DDCt 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. Brie y, the puri ed 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 plates were washed three times with PBS containing Tween-20 (PBST, pH 7.4) (5 min per wash), then incubated with 5% (w/v) skimmed milk powder at 37 °C for 90 min, then with100 μl of the two-fold gradient dilution of P. cuniculi-positive and negative serum samples (ranging from 1:20 to 1:320) were added and incubated at 37 °C for 1 h. The plates were 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., Wuhan, China). 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 Scienti c, 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 [2].
To further evaluate the feasibility of the iELISA, 50 P. cuniculi-positive serum samples were assessed by the iELISA, and the sensitivity was calculated as (ELISA positive × 100)/true P.cuniculi-positive [2]. 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-ve negative serum samples from naïve rabbits and 30 serum samples in the cross-activity assay were used to determine the speci city of the iELISA, which was calculated as (ELISA negative × 100)/real P. cuniculi-negative [2]. After that, the area under the receiver operating characteristic curve (AUC), a graph of the sensitivity (true positive rate) versus 1-speci city (false positive rate), was calculated by MedCalc 19.0.7 [17].
The repeatability (intra-assay variability) and reproducibility (inter-assay variability) of the iELISA were evaluated using three P. cuniculi-positive serum samples, substantially as previously described [18].
The experimental infestation of rabbits with P. cuniculi and serological testing using the established iELISA Rabbits infected with P. cuniculi were treated strictly as previously described [2]. Brie y, ten 3-month-old naive New Zealand rabbits (5 females and 5 males) were infested with P. cuniculi, and three non-infested rabbits were applied 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. cuniculi infestation rabbits and 15 from the noninfestation rabbits) were examined by the established optimal iELISA method. Each serum sample was tested in triplicate and analyzed in one ELISA plate, with positive and negative controls were contained in the plate.

Statistical analysis
All data are presented as the 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 signi cant.

Sequence analyses of two serpins
The 1572 bp open reading frame (ORF) in Pso c 27 cDNA (GenBank: MT707535) encodes 523 amino acids (aa), while 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 no signal peptide and a transmembrane region.

Expression and identi cation of two recombinant serpins
The 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 P. cuniculi-positive sera and the correspondent anti-serum IgG from rabbits, but not negative sera, revealing the favourable reactivity and antigenicity (Fig. 3).
Tissue localization of two serpins in adult female P. cuniculi Native Pso c 27 and PsoSP2 were located in ovary and mouthpart of female mites, respectively (Fig. 4b,  c). No uorescence signal was observed in adult female mites using pre-immunized rabbit IgG antibodies (Fig. 4a).

Serodiagnosis potential of two recombinant serpin proteins
By checkerboard titration, the optimal working conditions of 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 OD450 were 0.633 of rPso c 27 and 0.490 of rPsoSP2, respectively.
Serodiagnostic test of rabbits experimentally infested with P. cuniculi After 4 weeks of post-infestation (p.i.), all infested rabbits were observed with the visible ear scabs. Meanwhile, skin scrapings were positive for P. 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). The positive anti-rPso c 27 above the cut-off value was rst detected with 2/10 serum samples at 1-week p.i. in the infestation group. Afterwards, the rate of positive serum gradually increased to 80% (8/10) at 2 and 3 weeks p.i., then up to 100% (10/10) at 4 weeks p.i. (Fig. 8). In the non-infestation group, the anti-rPso c 27 antibody appeared below the cut-off value throughout the experiments.

Discussion
In the present study, two P. cuniculi serpins were characterized, and the potential of the recombinant proteins was evaluated for serodiagnosis of P. cuniculi infestation in rabbits. The predicted amino acid sequence showed the low overall identity of serpins compared to other mites, however, these two target proteins were identi ed as typical serpins due to the presence of the features such as serpin domain and serpin signature in C-terminal end [20]. Pso c 27 shared 50.85% amino acid sequence identity with the newly characterized D. farinae Der f 27 allergens, which has been proven to orchestrate the pulmonary in ammatory response and mediate Th2 type response in mice [21]. Besides, NJ analysis revealed that Pso c 27 yielded a close relationship with Der f 27. In combination with the homology and the genetic relationship between Pso c 27 and Der f 27, Pso c 27 may be considered as an allergen of P. cuniculi, which was possibly associated with the instigation of the host cutaneous pro-in ammatory response [22]. Additionally, this cutaneous in ammation resulted in serum extravasation to provide su cient food for mite population growth and cause aggravation of 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. cuniculi. However, signi cant differences were seen for the transcription of Pso c 27 in female mites, with the highest level of expression showing a 347-fold change. In addition, the native protein was located in the ovary of female mites, indicating that Pso c 27 possibly was essential in vitellogenesis [19,23]. This role of serpin being involved in vitellogenesis has been proven in a recent study, which indicated 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 host's complement attack. In this study, PsoSP2 showed homology to the S. scabiei serpin family genes (20.98-54.13 % amino acid sequence identity), some of which have been con rmed to inhibit the activation of complement pathways [25,26]. Moreover, the native PsoSP2 localized in the mouthpart of female mites and its cDNA expression throughout the life stages of mites suggested 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 the rabbit breeding industry in China [27]. Thus, timely diagnosis and treatment of P. cuniculi infestation in rabbits are of paramount importance to reduce the risk of disease transmission and improve pro tability. In China, the current microscopic diagnosis for this disease is extremely time-consuming and ine cient in the low mite carriers and sub-clinical infestations in rabbits. Thus, it is imperative to seek for the effective immunoreactive antigens for rapid and accurate diagnosis of P. cuniculi infestation in rabbits. Furthermore, animals infested with P. ovis could evoke sero-speci c antibody [1,28], and this sero-speci c antibody was induced at the early phase of parasite infestation when animals appeared asymptomatic [1,2,29]. Thus, the enzyme-linked immunosorbent assay (ELISA) can be considered as an accurate method in the detection the low mite carriers and/or sub-clinical infestations when compared with microscopy of skin scrapings. In a previous study, serpin of Schistosoma mansoni was considered as a promising species-speci c diagnostic antigen in human schistosomiasis [10]. Therefore, in this study, we evaluated the serodiagnostic potential of rPso c 27 and rPsoSP2 by the establishment of the iELISA.
Compared to rPsoSP2-iELISA, the rPso c 27-iELISA displayed better diagnostic e ciency with higher values of sensitivity, speci city and AUC (rPso c 27 -rPsoSP2: 96.0-90.0%; 90.91-78.18%; 0.988-0.964, respectively). Although rPso c 27 showed cross-reaction with sera from 3/10 S. scabiei-infestation, the cross-reaction between these two ectoparasites have been commonly shown in other studies [2,29,30]. Fortunately, these two mite species were effectively treated with the same acaricide [3,31]. Besides, 1/10 rabbits infested with S. scabiei, C. pisiformis and Eimeria spp. showed a sero-reaction with rPso c 27; however, their OD values were close to the cut-off value and appeared markedly lower than those rabbits infested with P. cuniculi (F (1, 78) = 115.444, P < 0.0001). Moreover, rPso c 27-iELISA can 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. Regarding the high sensitivity and speci city, Pso c 27 was more suitable as a candidate antigen for serodiagnosis of P. 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 with the regular serpin domain and signature. The gene expression of Pso c 27 and PsoSP2 were found in all life stages of mites, with signi cantly high expression in adult female mites. Compared to rPsoSP2, rPso c 27 seemed to display a better diagnostic e ciency than PsoSP2 by iELISA, suggesting that Pso c 27 could be developed as a potential antigen for serological diagnosis of P. cuniculi infestation in rabbits, especially at the early stage of infestation. Psoroptes ovis (PSOVI22g04610) and P. ovis (PSOVI22g04560) are obtained from the Online Resource for Community Annotation of Eukaryotes (OrcAE) (https://bioinformatics.psb.ugent.be/orcae/ overview/Psovi). Helices are marked as red tubes, and sheets as dark green arrows on the sequence. Elements of secondary structure are labelled as follows: (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 Figure 2 The neighbor-joining (NJ) tree was constructed based on the deduced amino acid sequence of serpin.

Declarations
The numbers at nodes are the bootstrapping frequency (Bf) values of 1000 replications  Immunolocalization of Pso c 27 and PsoSP2 in the adult female of Psoroptes cuniculi. a Incubated with the negative IgG of the rabbit before immunization. b Incubated with the speci c IgG of anti-rPso c 27. c Incubated with the speci c IgG of anti-rPsoSP2. All images were taken under a uorescent microscope at 100× magni cation.

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