Development of a rapid scabies immunodiagnostic assay based on transcriptomic analysis of Sarcoptes scabiei var. nyctereutis

Scabies is a highly contagious skin disease caused by the mite Sarcoptes scabiei that affects many mammals. However, the sensitivity of traditional tests for scabies diagnosis in humans is less than 50%. To simplify the diagnosis of scabies, methods that are simple, sensitive, specific, and cost-effective are required. We developed an immunodiagnostic test based on S. scabiei var. nyctereutis RNA-seq data collected from Japanese raccoon dogs with sarcoptic mange. Three candidate antigens—a highly expressed hypothetical protein “QR98_0091190,” another mite allergen known as “SMIPP-Cc,” and an abundant “vitellogenin-like protein”—were evaluated by western-blot analysis. A lateral flow immunoassay, using specific antibodies against the vitellogenin-like protein, successfully detected scabies in the skin flakes of S. scabiei-infected raccoon dogs. This assay can potentially diagnose scabies more accurately in wildlife, as well as in humans.

Sequencing and assembly. In total, 162,918 contigs, with a maximum length of 30,727 bp and a minimum length of 201 bp (N50 = 4,964 bp) were generated by de novo assembly (Table 1). Among these, 98.4% (109,185/110,911) were homologous to S. scabiei var. canis gene sequences and 110,033 were annotated. Data of Figure 1. Schematic overview of this study for the development of a lateral immunoassay for scabies diagnosis. This is an original diagram drawn by co-author Chiaki Sakuma and us. www.nature.com/scientificreports/ Transcriptional ranking. The selection of the most adequate molecules for development of a lateral flow immunoassay was based on gene expression levels. The top 50 highly expressed genes out of 162,918 in terms of transcripts-per-million (TPM) for each sample are depicted in Fig. 2 and Supplementary Table 1. The gene with the highest expression was that encoding the hypothetical protein QR98_0091190 (IACW01014455). This protein was ranked significantly higher than the other 14 hypothetical proteins. Two mitochondrial genes were ranked 9th and 10th. In addition, the ranking of seven genes of mitochondrial origin among those of the 50 genes was also distinctive (represented by the light blue highlight) (Supplementary Table 1). The hypothetical protein QR98_0091190 could be thought to be a candidate diagnostic target. However, the contig encoding this protein has several simple sequence repeats and may be considered as an artifact of the sequence assembly process. Therefore, we confirmed the sequence of the contig encoding QR98_0091190 by Sanger method, using PCR products obtained from total RNA by RT-PCR. A PCR product with an expected size was identified, and the nucleotide sequence of the PCR product was matched to the assembled contig (Supplementary Figure 1). Therefore, it has been confirmed that the assembled contig was correct.
Selection of candidate antigens. To select candidate antigens, we compared data obtained in this transcriptome study with GenBank data and public proteomic data available for S. scabies var canis 10 . We selected three antigens, a hypothetical protein QR98_0091190 (IACW01014455), vitellogenin-like protein (IACW01000323), and SMIPP-Cs (the Scabies Mite Inactivated Cysteine Protease Paralogs) (IACW01021976) ( Table 3). The reasons for the selection are as follows:  www.nature.com/scientificreports/ The hypothetical protein QR98_0091190 (theoretical molecular weight: 33 kDa) was ranked first in terms of gene expression levels in the newly obtained RNA-seq data (Fig. 2) and was a unique protein non-homologous to proteins from other organisms.
SMIPP-Cs (transcript ranked 104th) (molecular weight: 36.48 kDa) was the closest homolog to the group 1 allergens of house dust mites (HDMs) ( Table 2), which are proteolytic papain-like cysteine proteases that promote pathogenicity in asthma and allergy 22 (Supplementary Figure 2). It is also commonly used for a commercially available HDM allergy test. As it is a homolog of the HDM allergen 1 and easily induces antibody formation in the host, we hypothesized that SMIPP-Cc might also act as a stable protein with similar properties, such as retention in host tissue for a long time and ease of uptake by immune cells. Indeed, studies on human scabies mites have shown that SMIPP-C may interfere in the functions of host proteins present in the epidermis or interact with them 22,23 .
Vitellogenin-like protein (transcript ranked 28th) (molecular weight: 220.78 kDa) shared low identity with D. pteronyssinus vitellogenin (52%) and D. farinae vitellogenin (51%, our inference; refer to the subsection "Evaluation of antigens by western blotting" in detail.) in the BLAST search. Additionally, high protein expression of dog scabies mites was reported in proteome data 18 , with the highest number of spots (11/97 spots) observed in the 2D electrophoresis of aqueous extracts. Although Sar s 28 (heat shock protein 70-like protein; gene expression ranked 29th) and actin-like protein 6 (gene expression ranked 47th) were also shown to have 5/97 spots and 1/97 spots, respectively, in the proteomic data (Table 4), these two proteins were not selected as antigen candidates because of their high homology to proteins in other ticks (there is an 89.18% identity between Sar s 28 and D. pteronyssinus heat shock cognate 71 kDa protein and a 95.39% identity between actin-like protein 6 and Phynus mexicanus actin) ( Table 4).

Evaluation of antigens by western blotting.
Polyclonal antibodies were generated using the three candidate antigens by immunizing rabbits with the peptides synthesized by selecting amino acid sequences at positions with no or low homology compared to HDM proteins (Fig. 3, Supplementary Figures 2, 3, 4, and Supplementary Table 1). Note that D. farinae apolipophorin differed from the other vitellogenins in sequence as shown in the bottom row of Fig. 3. Therefore, we predicted novel vitellogenin-like sequences from scaffolds of the D. farinae whole genome and compared them to those of S. scabies (Fig. 3). Western blotting analysis using antibodies against the three antigens is depicted in Fig. 4 and Supplementary Figure 6. When anti-vitellogeninlike protein antibody was used, a specific and clear band at 17 kDa and a weak band at 70 kDa and 150 kDa were detected. There was no or low cross-reactivity with HDM proteins. In the case of the anti-hypothetical protein QR98_0091190 antibody, three clear bands were identified at 50, 17, and 10 kDa; a faint band at 30 kDa was also detected in D. farina, which indicated that this antibody weakly cross-reacted with HDM. Furthermore, the hypothetical protein QR98_0091190 antigen was found to be soluble in extraction solution containing sodium dodecyl sulfate (SDS) detergent but insoluble in physiological buffer without SDS (Supplementary Figure 5). Therefore, the solubility of this protein in aqueous solutions was low, making it unsuitable for use as a diagnostic antigen.  www.nature.com/scientificreports/  www.nature.com/scientificreports/ When anti-SMIPP-Cc antibody was used, strong signals were detected at 40 kDa and 60 kDa. Several bands were also observed for HDMs, confirming the cross-reactivity of this antibody (Fig. 4).
Vitellogenin-like protein was selected based on its high expression level in mites, low identity to HDM proteins, and its solubility, which makes easier the purification process.

Structural analysis of S. scabies vitellogenin-like protein. Domain architecture analysis using
Pfam revealed that the amino acid sequence of the S. scabies vitellogenin-like protein contains three conserved domains ( Table 2), and the epitope sequences among the three mite isolates were 100% identical (Fig. 3).
Phylogenetic analysis revealed that scabies mite vitellogenin is evolutionarily close to vitellogenin from two HDM species and sheep mite; however, the epitope sequence itself is unique to Sarcoptes, confirming that vitellogenin from scabies mite serves as a strong candidate as a diagnostic antigen for scabies (Fig. 5).
Lateral flow immunoassay. Based on the data of the solubility of the selected antigen in physiological buffers and the high specificity of the antibody against it, a device for lateral flow immunoassay (LFIA) using anti-vitellogenin polyclonal antibodies was developed (Fig. 6). The limit of detection (LOD) or analytical sensitivity of the LFIA was found to be between 0.1 and 0.2 ng/mL of vitellogenin protein evaluated by visual estimation or using an immunochromato reader after 15 min of migration when the synthesized peptide antigen was used (Fig. 6A). Further, positive results were obtained using ten individual skin extracts from scabies-infected raccoon dogs, with the formation of a clear band at the test line (Supplementary Figure 7). One healthy skin extract and one cured skin extract treated with ivermectin gave negative results. The intensities for the mange groups were significantly higher (P < 0.05) than those for the non-mange groups (Fig. 6B). The negative result obtained in the LFIA confirms the efficacy of the treatment in the ivermectin-treated racoon dog.

Discussion
Traditional tests to diagnose scabies in humans still have less than 50% accuracy 6,7 . To overcome this low sensitivity, we tried to develop an immunodiagnostic test for scabies mites based on biochemical and bioinformatic analysis. For the first time, RNA-seq data were obtained by the sequencing of mite RNA from scabies-infected wild raccoon dogs. The database of de novo RNA-seq of Sarcoptes scabiei var. canis 24 remained undisclosed and could not be used as a reference. We performed transcriptomic analysis and selected the candidate antigens for diagnosis based on gene expression levels, homology comparisons, number of allergens, and proteomic analysis using data from public databases.
Eventually, vitellogenin-like protein was selected as the diagnostic antigen as it is a soluble protein found in abundance in mite lysates, has low homology to the proteins in HDMs and other mite species, and its specific antibodies exhibit no or low cross-reactivity with HDM proteins. Moreover, western blot analysis revealed the existence of multiple molecules of vitellogenin in the scabies mite. In insects, vitellogenins are large molecules (~200-kD) synthesized in fat bodies through a process that involves substantial structural modification (e.g., glycosylation, lipidation, phosphorylation, and proteolytic cleavage, etc.) of the nascent protein prior to its secretion and transport to the ovaries 25 . Additionally, vitellogenin in ticks (Ixodes scapularis, Dermacentor variablis, Haemaphysalis longicornis) and Acari mites (Tetranychus urticae) contained multiple proteolytically cleavable subunits with different molecular weights [26][27][28][29] . Therefore, it is assumed that scabies mite vitellogenin also has multiple subunits that can be separated by cleavage with proteases. The specific antibodies developed herein were thought to strongly recognize the soluble form of the small, processed vitellogenin rather than the large precursor protein. This is considered to be advantageous in the lateral flow immunoassay (LFIA), wherein small and abundant antigen-antibody complexes flowed easily and efficiently across the surface of the nitrocellulose membrane. A second advantage of the developed LFIA is that the epitope sequence of vitellogenin in this study was 100% identical in mites that are parasitic to humans, dogs, and raccoon dogs, suggesting that the immunodiagnostics of scabies is feasible, not only in raccoon dogs but equally so in humans and other mammals. Another advantage of the developed LFIA is that the homology of vitellogenin with proteins from Acari mites and ticks related to scabies mites, as indicated in our studies, was 50-60%, which makes scabies diagnosis using our assay highly reliable, because LFIA is based on the use of a polyclonal antibody that does not cross-react with antigens from HDMs, possibly ticks, or other Acari mites. It has previously been reported that vitellogenin has an organism-specific amino acid sequence; for example, the expression of vitellogenin in fish has also been put to practical use as a biomarker to detect known endocrine-disrupting chemicals in contaminated environments 30 .
Thus, the LFIA method presented herein is a straightforward, rapid, sensitive, specific, and cost-effective test for the diagnosis of scabies. LFIA is portable and requires no special equipment, as opposed to PCR or ELISA. Additionally, the total cost of the materials used for the assay is typically a few dollars; hence, it could technically be performed at a cost that is 1/100th to 1/10th the cost of other testing methods, considering the capital www.nature.com/scientificreports/ expenditure on equipment. In addition, the antibodies produced in this study can be stored stably at −80 °C for several years, and the LFIA components can be refrigerated at 4 °C for 1-2 years. The prototype LFIA yielded 100% sensitivity and specificity when skin samples from raccoon dogs with confirmed scabies mange were tested. However, we only assessed a small number of samples in this study; thus, the assay developed herein remains a prototype at present.
Future studies should involve the assessment of more samples using appropriate negative controls to evaluate the feasibility of the developed assay as a diagnostic tool for scabies. Other candidate scabies mite antigen targets can be selected using the same RNA-seq data and public databases used by us and can be used to develop specific antibodies for more accurate, sensitive, and rapid diagnosis of scabies.
Our results provide new insights into the biology of scabies caused by Sarcoptes scabiei and new strategies for its diagnosis.

Methods
Animal study. All   Library preparation and next generation sequencing. An mRNA-seq library was prepared in accordance with Illumina's TruSeq RNA sample preparation protocol to minimize contamination from microbial RNA derived from symbiotic poly (A)-less bacteria. Briefly, mRNA was enriched from total RNA using oligo (dT)-attac hed magnetic beads after pretreatment of the products obtained from DNA digestion by DNase I. The enriched mRNA was broken into short fragments for first-strand cDNA (ss cDNA) synthesis, and, subsequently, second-strand cDNA (dsDNA) was synthesized and purified using the QIAquick PCR Purification Kit (Qiagen, Germany), accompanied by adaptor ligation. Finally, ds cDNA fragments of a suitable size were separated by agarose gel electrophoresis for PCR amplification. The prepared library was sequenced to obtain 100 bp pairedend reads using the HiSeq 2000 system (Illumina, CA). www.nature.com/scientificreports/ RNA-seq da ta processing. Raw read data were processed to remove adapter sequences from the 3′ ends, as well as nucleotides with low quality base calls (Quality Score < 30) from both the 5′ and 3′ ends of reads. The trimmed reads were discarded using Cutadapt version 1.9.1 if they contained more than five ambiguous bases (Ns) or if they were shorter than 20 bases 33 . Quality assessment of the raw and trimmed reads was performed using both Cutadapt and FastQC version 0.11.5 34 . The trimmed paired-end reads were assembled de novo using Trinity version 2.2.0 35 , and ORFs of the assembled transcripts were predicted using TransDecoder version 2.1.0 36 Functional annotation of the assembled transcripts and predicted proteins was performed using Trinotate version 3.0.1 37 . Furthermore, sequence similarity searches of the predicted coding sequences and peptide sequences were performed against the National Center for Biotechnology Information (NCBI) non-redundant nucleotide (nt) and protein (nr) sequence databases using BLASTn and BLASTp version 2.3.0 38 . The three peptides for immunization were selected based on a comprehensive analysis of antigenic site prediction using multiple algorithms (antigenicity, hydrophilicity/hydrophobicity, and secondary structure, among others).
Identification of potential allergens. To determine homology of allergen gene sequences between the presently acquired RNA-seq data and the draft genome data of dog mites 16 , including data for three mite species besides the scabies mite, 287 allergen genes from Dermatophagoides farinae, Dermatophagoides pteronyssinus, and Euroglyphus mayne were downloaded from NCBI (http:// www. ncbi. nlm. nih. gov/). Antibody preparation focused on the three peptides that were linked with a carrier protein (keyhole limpet hemocyanin). The resulting conjugate was homogenized using Freund's complete adjuvant for the initial injection (0.3 mg) at day 0 and with Freund's incomplete adjuvant for the booster injections (0.2 mg) at days 28, 35, and 42. The conjugate was then injected into the rabbits (Japanese white). On day 49, blood was drawn from the immunized rabbits and the antibodies were then harvested.

Ranking of gene expression.
Western blotting. The specificity of the three rabbit polyclonal antibodies was analyzed using western blotting. Scabies mites, collected using a method previously reported method 20 . Dermatophagoides farina mites (# bo002), and Dermatophagoides pteronyssinus (#ybo002) mites (Biostir, Hiroshima, Japan) were lysed using urea buffer (8 M urea, 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.1% Triton X-100) and then sonicated on ice for 1 min using a handheld sonicator (TOMY SEIKO, Tokyo, Japan). Whole lysates were mixed with loading buffer containing 1,4-dithiothreitol and heated at 95 °C for 5 min. Subsequently, the samples were subjected to 10-20% gradient SDS-PAGE. The proteins in the gel were transferred onto a polyvinylidene fluoride (PVDF) membrane (BIO-RAD) using a Trans-Blot Turbo Transfer System (BIO-RAD) at a constant current of 1.3 A for 7 min 43 . After blotting, the membrane was blocked using a PVDF Blocking Reagent for Can Get Signal (TOY-OBO, Tokyo, Japan) for 1 h and then treated with the primary rabbit antibodies diluted using Can Get Signal 1 (TOYOBO) (1:2000) at room temperature (20-25 °C) for 16 h; subsequently, it was treated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (#7474; Cell Signaling Technology, USA) in Trisbuffered saline supplemented with 0.1% Tween 20 at room temperature (20-25 °C) for 1 h. Antibody binding was detected using the enhanced chemiluminescence detection method with the SuperSignal West Femto PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and ImageQuant LAS500 analyzer (Cytiva, MA) or the colorimetric detection method using the 3,3′,5,5′-tetramethylbenzidine substrate AE-1490 EzWestBlue (Atto Corp, Japan).
Lateral flow immunoassay evaluation. An LFIA cassette for detecting the vitellogenin-like protein of scabies was manufactured by Kyokuto Pharmaceutical Industrial Co. Ltd (Takahagi, Japan) using a rabbit antiraccoon dog mite vitellogenin like protein polyclonal antibody. In the LFIA, polyclonal antibodies can detect antigens using a capture-and-detect antibody sandwich in the presence of multiple amino acid sequences in the synthetic peptide (30 mer) that the antibody recognizes. A rabbit polyclonal antibody was conjugated to colloidal gold. Briefly, for conjugation, 70 μg of rabbit polyclonal antibody dissolved in PBS (0.7 mg/mL) was added to 1 mL of colloidal gold solution. The mixture was stirred for 16 h at room temperature (20-25 °C), and then 10% (w/v) bovine serum albumin (BSA) solution was added. The labelled bioconjugates were centrifuged at 9,800 g and 4 °C for 15 min. The obtained precipitate was resuspended with 5 mM Tris-HCl (pH8.2) buffer, containing 150 mM NaCl, BSA (1%, w/v), and sucrose (5%, w/v). The final product was stored at 4 °C in the dark for further use. The detection zone contained immobilized goat anti-rabbit antibodies as a control line and non-labeled www.nature.com/scientificreports/ anti-vitellogenin-like protein rabbit polyclonal antibody as a test line on nitrocellulose membranes. A visible control line indicated that the gold-labeled antibody flowed along the test strip and performed appropriately. We studied both relatively ordinary/common mange and crusted mange. For common mange, 5 mm of the skin containing hair was collected from the carcass of raccoon dogs immediately after their death. For crusted mange, a crusted hairless skin sample was manually collected from the raccoon dog's body without inflicting pain. The sample was suspended in 500 μL of extraction buffer (lysis step, SDS solution) for a few seconds. Subsequently, the extract was diluted 100 times using the assay solution (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween 20), following which 100 μL was dispensed onto the cassette. The results were visually evaluated or determined using the Immunochromato reader C10066-10 (Hamamatsu photonics, Hamamatsu, Japan) and after 15 min of migration by monitoring the appearance of a red band specific to vitellogenin, along with a band corresponding to the internal control. Pictures of the Immunochromato results were taken using TCR-500 Immuno reader (Trust-Medical, Kasai, Japan) and digital camera. The Tukey test was performed to evaluate statistical significance of results.
Determination of the analytical sensitivity of the LFIA. Serial dilutions (4, 2, 0.4, 0.2, 0.1, and 0 ng/ ml) of the vitellogenin-like derived synthesized peptide (amino acids KWSAETRTNNLRQIARQAAQEEAAR-QQQM) were prepared using the extraction buffer. Subsequently, 100 μl of each diluted solution was dispensed onto the cassette and allowed to migrate for 15 min. Results were visually evaluated using the Immunochromato reader.
Sequence comparisons and phylogenetic analysis of vitellogenin. The putative amino acid sequences of the vitellogenin-like protein of S. scabiei and other arthropods were obtained from GenBank and aligned using ClustalX version 2.0 44 . The sequence similarities were analyzed using the online BLASTP program available on the NCBI website (https:// blast. ncbi. nlm. nih. gov/). The molecular weights and isoelectric points (pIs) of the deduced protein based on the sequences obtained were predicted using the ExPASy proteomics server (http:// www. expasy. org). The domain architecture and conserved domains were analyzed using the online servers of Pfam (http:// pfam. xfam. org/). The signal peptide was predicted using the SignalP 4.1 server (http:// www. cbs. dtu. dk/ servi ces/ Signa lP). The potential O-linked glycosylation sites were predicted using the GPP Prediction Server (http:// comp. chem. notti ngham. ac. uk/ glyco/). The proprotein convertase cleavage sites were predicted using the ProP 1.0 Server (http:// www. cbs. dtu. dk/ servi ces/ ProP/).
Phylogenetic and molecular evolutionary analyses were conducted using MEGA X 45 . A phylogenetic tree was constructed using the neighbor-joining method 46 . The evolutionary distances were computed using the Poisson correction method 47 and are represented as the number of amino acid substitutions per site. All ambiguous positions were removed for each sequence pair (pairwise deletion option). The statistical confidence of a particular cluster of sequences was evaluated using the bootstrap test with 500 replications 48 . Furthermore, since the amino acid sequence of D. farina apolipophorin (BBD75204.1) was found to be significantly less homologous than the vitellogenin amino acid sequence, a new homolog was predicted from the D. pteronyssinus draft genome data 49 . Moreover, a vitellogenin homolog of the closely related mite Psoroptes ovis was inferred from recent whole-genome data 50 . Ethics declaration. This research adheres to the "Japanese Association of Zoos and Aquariums Ethics and Welfare Guidelines" and "Caring for Wildlife: The World Zoo and Aquarium Animal Welfare Strategy", and was approved by the Kanazawa Zoological Gardens ethical and welfare assessors, Permit Number: #RKK1111 Kanazawa Zoological Gardens, February 20, 2015. All the animal experiments were conducted in compliance with the ARRIVE guidelines. S. scabiei mites were collected from the crusted skin naturally shed by a single infected wild raccoon dog without sufferring of the animal involved.