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

Abstract

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.

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.

Figure 2

Transcript abundances of annotated genes in the S. scabies transcriptome. Top 50 genes ranked according to expression level in terms of transcripts-per-million (TPM) for each sample are shown as a barplot.

Figure 3

Conserved domain architecture of vitellogenin-like protein in S. scabiei and alignment of the sequences of proteins from the three types of scabies mites and two types of house dust mites. (A) The vitellogenin N (lipoprotein amino terminal region), DUF1943 (domain of unknown function), and VWD (von Willebrand factor type D domain) domains were predicted based on significant Pfam matches. (B) Comparison of sequences proximal to the selected peptide sequence in the vitellogenin-like proteins (red line) from S. scabiei, D. farinae (BBD75204.1), and D. pteronyssinus (XP_027195573.1). With respect to the amino acid sequence of the peptide used for immunization, the sequence homology was 45%–48% between S. scabiei and HDM and 83% between D. pteronyssinus and D. farinae. Boldfaced text or the use of an asterisk indicates perfect alignment; the dot indicates weak similarity.

Figure 4

Western blot analysis of the three target antigens for S. scabies diagnosis. The whole mite lysates (20 μg/lane) of S. scabies and HDMs (D. farina, D. pteronyssinus) were immunoblotted with preparations of anti- hypothetical protein QR98_0091190, SMIPP-Cc, and vitellogenin-like protein rabbit polyclonal antibody. Lane 1, molecular weight marker; Lane 2, whole lysate of S. scabies (Ss); Lane 3, whole lysate of D. farina (Df); Lane 4, whole lysate of D. pteronyssinus (Dp); The red arrows indicate the specific bands (17, 70, and 150 kDa) exhibiting reaction to the anti-vitellogenin-like protein antibody. The black arrows indicate the three clear bands (50, 17, and 10 kDa) in the experiment in which the anti-hypothetical protein QR98_0091190 antibody was used and the two bands (40 and 60 kDa) detected using the anti-SMIPP-Cc antibody. These photo data were taken at the same exposure time (4.7 s).

Figure 5

A molecular phylogenetic tree of arthropod vitellogenin was constructed. The DDBJ/GenBank/EMBL accession numbers are follows: IACW01000323 (Sarcoptes scabiei var nyctereutis Vg like protein), KPM02263.1 (Sarcoptes scabiei var canis Vg like protein), Creation by scaffold41081_cov61 and 6362_cov109 (Sarcoptes scabiei var hominis Vg like protein), XP_027195573.1 (Dermatophagoides pteronyssinus Vg6), Creation by scaffold NBAF01001615.1 (Dermatophagoides farina Vg), Creation by scaffold PQWQ01000014.1 (Psoroptes ovis Vg), RWS06019.1 (Dinothrombium tinctorium Vg), RWS27858.1 (Leptotrombidium delicense Vg), AHN48900.1 (Panonychus citiri Vg), AYV89292.1 (Tetranchus evansi Vg4), AMO02571.1(Tetranchus cinnabarinus Vg4), AMO02572.1 (Tetranchus cinnabarinus Vg6), AYV88983.1 (Tetranchus truncates Vg), XP015793836.1 (Tetranchus urticae Vg6), XP022244466.1 (Limulus polyphermus Vg3), XP023225368.1 (Centruroides sculpturatus Vg5), AXN69712.1 (Pardosa pseudoannulata Vg1), XP015930209.1 (Parateatpda tepidariorum Vg4X1), XP015930210.1(Parateatpda tepidariorum Vg4X2), XP022657753.1 (Varroa destructor Vg3), ABW82681.2 (Dermacentor variabliis Vg 2), BAH02666.2 (Ornithodoros moubata Vg), AFN88464.1 (Varroa destructor Vg2), XP022660223.1 (Varroa destructor Vg6), XP022701897.1 (Varroa jacobsoni Vg6), XP018494463.1 (Galendromus occidentalis Vg4), QCX36526.1 (Euseius nicholsi Vg), ASB34116.1 (Neoseiulus barkeri Vg2), AGQ56699.1 (Neoseiulus cucumeris Vg2), QBZ96191.1 (Amblyseius eharai Vg), AJR36491.1 (Ixodes ricinus hemolipoglyco-carrier protein CP3), ABD83654.1 (Dermacentor variabilis hemelipoglycoportein precursor), QEL09188.1 (Rhipicephalus haemaphysaloides Vg2), BAG12081.1 (Haemaphysalis longicomis Vg2), AXP34688.1 (Haemaphysalis flava Vg2), BBD75204.1 (Dermatophagoides farina apolipophorin), and BAA06397.1 (Bombyx mori Vg).

Figure 6

Lateral flow immunoassay for scabies vitellogenin-like protein. (A) Sensitivity of immunochromatography test, determined using standard vitellogenin-like protein. A total of 100 μl of serial dilutions (4, 2, 0.4, 0.2, 0.1, and 0 ng/ml) of the synthesized vitellogenin peptide antigen was dispensed onto the cassette and allowed to migrate. Results were read by eye or using an Immunochromato reader after 15 min. Milli Absorbance (mABS) indicated at the top of the lane. The limit of detection (LOD) is indicated using an arrow (0.1–0.2 ng/mL). (B) Evaluation of scabies diagnosis using the rapid lateral immunoassay. A total of 100 μL of lysate from skin scrapings of raccoon dogs or whole mite extracts of HDM was dispensed on to the cassette and allowed to migrate. Results determined using the Immunochromato reader are depicted as a dot-plot. It was possible to distinguish between positive (n = 10) and negative (n = 12) readings at a reader value of approximately 10. *; P < 0.05 skin scrapings of raccoon dogs