150 Vol. 6 No. 6 September–December 2017 Research Report MOLECULAR IDENTIFICATION OF SARCOPTES SCABIEI VAR. CUNICULI FROM SURABAYA AND MALANG REGIONS OF EAST JAVA Kurnia Desiandura1, nunuk Dyah Retno Lastuti2a, Lucia Tri suwanti2,4, Didik Handijatno3 1 Faculty of Veterinary Medicine, Universitas Airlangga 2 Department of Parasitology Veteriner, Faculty of Veterinary Medicine, Universitas Airlangga 3 Department of Microbiology Veteriner, Faculty of Veterinary Medicine, Universitas Airlangga 4 Institute of Tropical Disease, Universitas Airlangga a Coresponding author: nunuk_dyah@yahoo.com abstract Scabies is a zoonotic skin disease caused by Sarcoptes scabiei mites. As an emerging/re-emerging parasitic disease, scabies represents a significant global threat to both human and animal health. Numerous cases of scabies in Indonesia have been reported, which support research on the prevalence of S. scabiei. However, most such studies have involved conventional morphological studies, with limited molecular diagnostic studies. The purpose of the present study was the genetic characterization of S. scabiei var. cuniculi in domestic rabbits to generate baseline genotypic data. S. scabiei var. cuniculi was isolated and identified from scabies-infected rabbits from the Surabaya and Malang regions of East Java. Molecular identification was performed using Polymerase Chain Reaction (PCR) using specific primers targeting the COX1 gene. We performed COX1 PCR using rabbit isolates of S. scabiei from Indonesia. To the best of our knowledge, no such study had been reported previously. This study was performed in the Laboratory of Veterinary Parasitology, Faculty of Veterinary Medicine and the Tropical Disease Diagnostic Center Laboratory, Universitas Airlangga. The results with agarose gel electrophoresis revealed a 289 bp PCR product amplified from the DNA of S. scabiei isolates from both Surabaya and Malang in accordance with the expected COX1 amplicon size, that indicated a single band 289 bp in length, demonstrating specific detection of S. scabiei var. cuniculi from Surabaya and Malang using COX1 primers. The results were consistent with the calculated amplicon size based on primer positions within the COX1 locus, with the forward primer spanning nucleotides 61–94, and the reverse primer spanning nucleotides 331–350 ( 350 − 61 = 289 bp). PCR genotyping of the isolates yielded an identical nucleotide length of 289 bp. Further studies are required to sequence the amplified fragments for homology assessment. Keywords: Sarcoptes scabiei, scabies, PCR, 289 bp, zoonosis abstrak Scabies disebabkan oleh tungau Sarcoptes scabiei adalah tungau penyebab penyakit kulit yang bersifat zoonosis. Scabies sebagai penyakit parasite yang muncul kembali dan mengancam kesehatan manusia maupun hewan di dunia. Kasus scabies yang tinggi pada beberapa hewan domestic seperti kelinci di Indonesia hanya didukung dengan hasil penelitian prevalensi S.scabiei, namun hanya bedasarkan tinjauan gejala klinis dan morphologi, sedangkan penelitian berbasis molecular seperti karakterisasi genetic dari tungau S.scabiei masih terbatas. Tujuan dari penelitian ini untuk mendeteksi karakterisasi genetik tungau Sarcoptes scabiei var.cuniculi dari kelinci domestik sebagai data dasar informasi genetik. Penelitian ini dilakukan isolasi dan identifikasi S.scabiei var.cuniculi dari kelinci yang menunjukkan gejala scabies dari daerah Surabaya dan Malang, selanjutnya dilakukan ektraksi DNA dari isolate S.scabiei kelinci Indonesia, untuk diproses dengan Polymerase Chain Reaction (PCR) menggunakan primer spesifik dengan target gen COX1 289 bp. Studi ini dilakukan di Laboratorium Parasitology Veteriner, Fakultas Kedokteran Hewan dan Laboratorium Pusat Diagnostik Penyakit Tropik, Universitas Airlangga. Hasil studi genetic S.scabiei isolate lokal Indonesia untuk menambah ilmu pengetahuan karena belum ada hasil studi yang dilaporkan sebelumnya. Hasil electrophoresis tungau S.scabiei var.cuniculi dari kelinci asal Surabaya dan Malang menunjukkan panjang nukleotida sebesar 289 bp sesuai dengan target. Hasilnya konsisten dengan ukuran amplikon yang 151Desiandura, et al.: Molecular Identification of Sarcoptes scabiei dihitung berdasarkan posisi primer di dalam lokus COX1, dengan primer ke depan mencakup nukleotida 61-94, dan primer terbalik mencakup nukleotida 331-350 (350 - 61 = 289 bp). Genotipe PCR dari isolat lokal menghasilkan panjang nukleotida identik 289 bp. Diperlukan penelitian lebih lanjut untuk mengurutkan fragmen yang diperkuat untuk penilaian homologi. Kata kunci: Sarcoptes scabiei, scabies, PCR, 289 bp, zoonosis introduction scabies or mange is an infectious zoonotic skin disease caused by the mite Sarcoptes scabiei and is considered an important disease in both humans and animals. It is estimated that more than 300 million people are infected each year.1,2 The sarcoptes mite is an obligate parasite of the skin; the mites burrow into the stratum corneum complete their life cycle starting from the egg to the adult stage.2–4 S. scabiei infection may trigger multiple reactions including allergic reactions, inflammation, innate immune reactions, and activation of immune components in the skin.5 significant clinical symptoms of scabies include thickening of skin, crust formation, alopecia involving the eyes, ears, mouth, legs, and itching accompanied by the formation of red spots (rash). Occasionally, 3-cm long lines or grooves are formed on the skin, which lead to crust, papule, or vesicle formation.6–8 scabies mainly occurs in areas with high poverty rates and low nutritional status and is transmitted via direct contact with infected humans or animals.9 Numerous cases of scabies in Indonesia have been reported, which support research on the prevalence of S. scabiei. However, most such studies have involved conventional morphological studies, with limited molecular diagnostic studies.10,11 Esther et al. (2015) reported PCR based detection of genotypic differences between S. scabiei from different hosts including pigs, rabbits, foxes, jackals, and porcupines in comparison with the Genbank database using three different genes, including cytochrome C oxidase 1 (COX1) with a 467 bp amplicon and Glutathione-s- Transferase class 1 (GST1) with a 670 bp amplicon. The highest percentage identity of database-reported sequences, at 99%, was with S. scabiei from rabbits, obtained using COX1 PCR. Another study reported an S. scabiei COX1 amplicon of 250 bp in an isolate from Hong Kong.11 The purpose of this study was the genetic characterization of S. scabiei var. cuniculi from domestic rabbits to generate baseline genotypic data. We performed COX1 PCR using rabbit isolates of S. scabiei from Indonesia. To the best of our knowledge, no such study had been reported previously. material and method S. scabiei mites (adults, nymphs, and larvae) were isolated from domestic rabbits from two different regions in East Java, including four rabbits from surabaya and two from Malang. Domestic rabbits showed clinical signs of scabies such as thickening of skin, crust formation, and alopecia involving the eyes, ears, mouth, and legs. Mites were morphologically identified using identification keys.6,7 This study was performed in the Laboratory of Veterinary Parasitology , Faculty of Veterinary Medicine and the Tropical Disease Diagnostic Center Laboratory, Universitas Airlangga. pcR Based Detection of s. scabiei in Rabbits DNA extraction was performed using mini kit QIAamp DNA (Qiagen, Hilden, Germany), following the manufacturer’s protocol as follows: 20 µl of Qiagen protease was added to a microcentrifuge tube with a 200 µl suspension of S. scabiei; 180 µl of buffer tissue lysis (ATL buffer) was then added and the tube was centrifuged at 8,000 rpm for 3 min, vortexed for 15 s, and incubated at 56°C for 24 h. Further, 200 µl of buffer lysis (AL buffer) was added to the tube and vortexed for 15 s; 200 µl of 96% ethanol was added to the tube, vortexed for 15 s, and centrifuged at 8,000 rpm for 1 min to pellet DNA. Next, 500 µl of buffer washing 1 (AW1 buffer) was added to the pellet and centrifuged at 8000 rpm for 1 min; 500 µl of AW2 buffer was then added to the pellet and the tube was centrifuged at 13,000 rpm for 3 min, at 13,000 rpm for 1 min, and 50 µl of elution buffer (AE buffer) was added to the final DNA pellet; following an incubation at 15–25°C for 1 min, the contents were centrifuged at 8,000 rpm for 1 min to collect DNA.12 Next, PCR to amplify a 289 bp region of the COX1 gene was performed using the forward primer 5’- TCTTAGGGGCTGGATTTAGTATG-3’ and the reverse primer 5’-AGTTCCTCTACCAGTTCCAC-3’. PCR was performed in an automatic thermocycler (Biorad) with an initial denaturation step at 95°C for 5 min, and 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by a final extension step at 72°C for 5 min.13 The PCR reaction product was then resolved using 2% agarose gel electrophoresis. result and discussion S. scabiei var. cuniculi mites were isolated from rabbits that showed clinical signs of scabies including thickening of the skin, crust formation, alopecia involving the eyes, ears, mouth, and legs. Morphological appearance of mites is spherical, transparent, and oval.6,7 The dorsal surface of their body contains fine-grooved lines equipped with a plastron, conical scales, and spines. They have four pairs 152 Indonesian Journal of Tropical and Infectious Disease, Vol. 6 No. 6 September–December 2017: 150–153 of short legs; two pairs of legs on the anterior (notothorax) with a sucker and ambulacra, and two pairs of posterior legs (notogaster). The mouth consists of a chelicera, capitulum, and hypostome 6 (Figure 1). Agarose gel electrophoresis revealed a 289 bp PCR product amplified from the DNA of S. scabiei isolates from both surabaya and Malang in accordance with the expected COX1 amplicon size. PCR reactions from both samples showed a band between the 200 bp and 300 bp molecular weight markers. The results were consistent with the calculated amplicon size based on primer positions within the COX1 locus, with the forward primer spanning nucleotides 61–94, and the reverse primer spanning nucleotides 331–350 ( 350 − 61 = 289 bp). Electrophoresis results are shown in Figure 2 and Figure 3. Phylogenetic studies on S. scabiei from rabbits using the COX1 sequence are limited; Wong et al. (2005) reported that the COX1 amplicon size using S. scabiei var. cuniculi isolated from Hong Kong was 250 bp, while that using S. scabiei var. cuniculi isolated from Indonesia, specifically Malang and surabaya, was 289 bp. Zhao et al. (2015) reported that it is unclear whether the same species of sarcoptes mites parasitize humans and animals, and data on genetic diversity of these mite populations in humans is scarce.14 In a study conducted in China to identify S. hominis and S. canis, genomic DNA was extracted from ten individual mites (five isolated from patients with scabies and five from dogs with mange) for amplification of ITs2 rDNA, 16s mtDNA, and COX1 fragment sequences. Future studies assessing the sequence of PCR products from S. scabiei are required to determine the level of homology among S. scabiei var. cuniculi originating from different countries, which can aid the development of subunit vaccines. scabies poses a significant threat to public health and causes economic loss because of high prevalence and the lack of efficient prevention measures. Dissatisfaction with use of insecticides is increasing because of the emergence of resistant parasites, environmental pollution, and consumer rejection of livestock products containing pesticide residues.15 Vaccination is the best available alternative because it causes least damage to the environment and is safe for consumer use, and potentially more effective than scabicide and inexpensive; however, several obstacles hinder large scale vaccine production. Therefore, several studies have been aimed at developing molecular subunit vaccines using recombinant DNA cloned from S. scabiei.16–19 8000 rpm for 1 min to pellet DNA. Next, 500 µl of buffer washing 1 (AW1 buffer) was added to the pellet and centrifuged at 8000 rpm for 1 min; 500 µl of AW2 buffer was then added to the pellet and the tube was centrifuged at 13000 rpm for 3 min, at 13000 rpm for 1 min, and 50 µl of elution buffer (AE buffer) was added to the final DNA pellet; following an incubation at 15–25 °C for 1 min, the contents were centrifuged at 8000 rpm for 1 min to collect DNA.12 Next, PCR to amplify a 289 bp region of the COX1 gene was performed using the forward primer 5'- TCTTAGGGGCTGGATTTAGTATG-3' and the reverse primer 5'- AGTTCCTCTACCAGTTCCAC-3'. PCR was performed in an automatic thermocycler (Biorad) with an initial denaturation step at 95 °C for 5 min, and 35 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by a final extension step at 72 °C for 5 min.13 The PCR reaction product was then resolved using 2% agarose gel electrophoresis. RESULT AND DISCUSSION S. scabiei var. cuniculi mites were isolated from rabbits that showed clinical signs of scabies including thickening of the skin, crust formation, alopecia involving the eyes, ears, mouth, and legs. Morphological appearance of mites is spherical, transparent, and oval.6,7 The dorsal surface of their body contains fine-grooved lines equipped with a plastron, conical scales, and spines. They have four pairs of short legs; two pairs of legs on the anterior (notothorax) with a sucker and ambulacra, and two pairs of posterior legs (notogaster). The mouth consists of a chelicera, capitulum, and hypostome 6 (Figure 1). Figure 1. Sarcoptes scabiei ventral view (Olympus Optilab Camera Microscope, Magnification: 400×) Agarose gel electrophoresis revealed a 289 bp PCR product amplified from the DNA of S. scabiei isolates from both Surabaya and Malang in accordance with the expected COX1 amplicon size. PCR reactions from both samples showed a band between the 200 bp and 300 bp molecular weight markers. The results were consistent with the calculated amplicon size based on primer positions within the COX1 locus, with the forward primer spanning nucleotides 61–94, and the reverse primer spanning nucleotides 331–350 ( 350 − 61 = 289 bp). Electrophoresis results are shown in Figure 2 and Figure 3. Hypostome Legs Bristle figure 1. Sarcoptes scabiei ventral view (Olympus Optilab Camera Microscope, Magnification: 400×) Figure 2. Agarose gel electrophoresis of the PCR product from S. scabiei var. cuniculi isolated from Surabaya Figure 3. Agarose gel electrophoresis of the PCR product from S. scabiei var. cuniculi isolated from Malang Phylogenetic studies on S. scabiei from rabbits using the COX1 sequence are limited; Wong et al. (2005) reported that the COX1 amplicon size using S. scabiei var. cuniculi isolated from Hong Kong was 250 bp, while that using S. scabiei var. cuniculi isolated from Indonesia, specifically Malang and Surabaya, was 289 bp. Zhao et al. (2015) reported that it is unclear whether the same species of Sarcoptes mites parasitize humans and animals, and data on genetic diversity of these mite populations in humans is scarce.14 In a study conducted in China to identify S. hominis and S. canis, genomic DNA was extracted from ten individual mites (five isolated from patients with scabies and five from dogs with mange) for amplification of ITS2 rDNA, 16S mtDNA, and 300bp 200bp 100bp 289bp M S 300bp 200bp 100bp 289bp S Information: M = Marker S = Sample Information: M = Marker s = sample figure 2. Agarose gel electrophoresis of the PCR product from S. scabiei var. cuniculi isolated from surabaya Figure 2. Agarose gel electrophoresis of the PCR product from S. scabiei var. cuniculi isolated from Surabaya Figure 3. Agarose gel electrophoresis of the PCR product from S. scabiei var. cuniculi isolated from Malang Phylogenetic studies on S. scabiei from rabbits using the COX1 sequence are limited; Wong et al. (2005) reported that the COX1 amplicon size using S. scabiei var. cuniculi isolated from Hong Kong was 250 bp, while that using S. scabiei var. cuniculi isolated from Indonesia, specifically Malang and Surabaya, was 289 bp. Zhao et al. (2015) reported that it is unclear whether the same species of Sarcoptes mites parasitize humans and animals, and data on genetic diversity of these mite populations in humans is scarce.14 In a study conducted in China to identify S. hominis and S. canis, genomic DNA was extracted from ten individual mites (five isolated from patients with scabies and five from dogs with mange) for amplification of ITS2 rDNA, 16S mtDNA, and 300bp 200bp 100bp 289bp M S 300bp 200bp 100bp 289bp S Information: M = Marker S = Sample figure 3. Agarose gel electrophoresis of the PCR product from S. scabiei var. cuniculi isolated from Malang 153Desiandura, et al.: Molecular Identification of Sarcoptes scabiei conclusion Sarcoptes scabiei isolated from rabbits that were infected with scabies was detected using COX1 PCR with the forward primer 5’-TCTTAGGGGCTGGTATTAGTATG-3’ and the reverse primer 5’-AGTTCCTCTACCAGTTCCAC-3’, yielding the expected amplicon size of 289 bp. references 1. Arlian LG, Vyszenski-Moher DL, Pole MJ. survival of adults and developmental stages ofsarcoptes scabiei var.canis when off the host. Exp Appl Acarol. 1989 Apr;6(3):181–7. 2. Morgan Ms, Rider sD, Arlian LG. Identification of antigenic sarcoptes scabiei proteins for use in a diagnostic test and of non- antigenic proteins that may be immunomodulatory. Vinetz JM, editor. PLos Negl Trop Dis. 2017 Jun 12;11(6):e0005669. 3. Walton sF, Currie BJ. Problems in Diagnosing scabies, a Global Disease in Human and Animal Populations. Clin Microbiol Rev. 2007 Apr 1;20(2):268–79. 4. Lastuti ND. specific antigenic protein 57.3 kDa of sarcoptes scabiei var. caprae as material candidate of scabies diagnostic kit for goat and Toll-like receptor mediated Immune Responses. Universitas Airlangga; 5. Erster O, Roth A, Pozzi Ps, Bouznach A, shkap V. First detection of sarcoptes scabiei from domesticated pig (sus scrofa) and genetic characterization of s. scabiei from pet, farm and wild hosts in Israel. Exp Appl Acarol. 2015 Aug 23;66(4):605–12. 6. soulsby EJL. Helminths, Arthropods and Protozoa of Domesticated Animals. 6th ed. London: E.L.B.s.; 1978. 7. Lastuti ND. Exploration of whole proteins sarcoptes scabiei var. cuniculi cause of rabbit’s scabies. Media Kedokteran Hewan. 2008;80–5. 8. Veteriner BP. Masalah skabies pada hewan dan manusia serta penanggulangannya. 1998;28–34. 9. Wardhana AH, Manurung J. skabies : tantangan penyakit zoonosis masa kini . dan masa datang. Ivartazoa. 2002;Vol. 16 No(30): 40–52. 10. Choy JL, Currie B, Arlian L, McBroom J, Kemp DJ, Bonson A, et al. Genetically distinct dog-derived and human-derived sarcoptes scabiei in scabies-endemic communities in northern Australia. Am J Trop Med Hyg. 1999 Oct 1;61(4):542–7. 11. Wong ssY, Poon RWs, Chau s, Wong sCY, To KKW, Cheng VCC, et al. Development of Conventional and Real-Time Quantitative PCR Assays for Diagnosis and Monitoring of scabies. Gilligan PH, editor. J Clin Microbiol. 2015 Jul;53(7):2095–102. 12. Reed R, Holmes D, Weyers J, Jones A. Practical skills in Biomolecular sciences (2nd Edition). second. Prentice Hall; 2003. 237-319 p. 13. Rantam FA. Methods of Immunology. surabaya: Airlangga University Press; 2003. 149-155 p. 14. Andriantsoanirina V, Ariey F, Izri A, Bernigaud C, Fang F, Charrel R, et al. sarcoptes scabiei mites in humans are distributed into three genetically distinct clades. Clin Microbiol Infect. 2015 Dec;21(12):1107–14. 15. TARIGAN s. Vaksin skabies dibutuhkan namun sulit diwujudkan. Wartazoa. 2007;17(30):38–45. 16. Rider sD, Morgan Ms, Arlian LG. Draft genome of the scabies mite. Parasit Vectors. 2015 Dec 10;8(1):585. 17. Arlian LG, Morgan Ms, Rider sD. sarcoptes scabiei: genomics to proteomics to biology. Parasit Vectors. 2016 Dec 1;9(1):380. 18. Zhang R, Jise Q, Zheng W, Ren Y, Nong X, Wu X, et al. Characterization and evaluation of a sarcoptes scabiei allergen as a candidate vaccine. Parasites and Vectors. 2012;5(1):1–9. 19. Casais R, Granda V, Balseiro A, del Cerro A, Dalton KP, González R, et al. Vaccination of rabbits with immunodominant antigens from sarcoptes scabiei induced high levels of humoral responses and pro-inflammatory cytokines but confers limited protection. Parasit Vectors. 2016 Dec 8;9(1):435.