Ehrlichia spp. disülfid oksidoredüktaz genini hedefleyen polimeraz zincir reaksiyonunun analitik duyarlılığının belirlenmesi: Klinik olarak leishmaniasis'ten şüphelenilen bir köpekte ehrlichiosis'in moleküler tanısı

Abstract

Ehrlichia spp. insan ve hayvanları enfekte edebilen kene kaynaklı zoonotik patojenlerdir. Günümüzde hastalığın tanısında kullanılan testler arasında yüksek duyarlılık ve özgünlüğe sahip olan moleküler yöntemlerin önemi giderek artmaktadır. Bu çalışmanın amacı Ehrlichia spp. disülfid oksiredüktaz (DSB) genini hedefleyen konvansiyonel polimeraz zincir reaksiyonunun (PZR) analitik hassasiyetinin belirlenmesidir. Öncelikle Ehrlichia spp. DSB geni TOPO vektörüne klonlanmıştır. DSB geni içeren TOPO plazmidi seri olarak sulandırıldıktan sonra Ehrlichia spp. DSB genini hedefleyen PCR gerçekleştirilmiştir. Bu araştırma üzerinde çalışmalar sürerken, klinik olarak leishmaniasis şüphesi bulunan ve leishmaniasis tedavisi alan bir sokak köpeğinin kan ve deri kazıntı örnekleri laboratuvarımıza gelmiştir. Sokak köpeğine ait kan ve deri kazıntı örneklerinde patojeni belirlemek amacıyla Ehrlichia spp. DSB ve 16S rRNA ile Leishmania spp. kinetoplast DNA (kDNA) genlerini hedef alan PCR testleri yapılmıştır. DSB geni içeren seri sulandırılmış TOPO plazmidi kullanılarak yapılan Ehrlichia spp. DSB genini hedefleyen PCR testinin analitik hassasiyeti, 1 ≥ kopya plazmit/reaksiyon olarak tespit edilmiştir. Klinik olarak leishmaniasis şüphesi bulunan sokak köpeğinin kan ve deri kazıntı örneklerinde Ehrlichia spp. DSB genini hedefleyen PCR pozitif, Leishmania spp. kDNA'yı hedefleyen PCR testleri negatif olarak tespit edilmiştir. Ehrlichia spp. 16S rRNA'yı hedefleyen nested PCR testi ile, sokak köpeğinin kan ve deri kazıntı örneklerinde E. canis olarak tanımlanmıştır. Bu çalışmada Ehrlichia spp. DSB genini hedefleyen PCR'nin yüksek duyarlılığa sahip olduğu gösterilmiştir. Ayrıca moleküler yöntemlerin klinisyenlere ehrlichiosis ve leishmaniasis ayırıcı tanısında uygunsuz tedaviyi önlemede yardımcı olabileceği öngörülmüştür.

Keywords

• Disülfit oksiredüktaz
• Ehrlichia canis
• Ehrlichiosis
• PCR
• Leishmaniasis

Determining the analytical sensitivity of polymerase chain reaction targeting Ehrlichia spp. disulfide oxidoreductase gene: Molecular diagnosis of ehrlichiosis in a dog clinically suspected with leishmaniasis

Abstract

Ehrlichia spp. is tick-borne zoonotic pathogen that can infect humans and animals. Nowadays, among the tests used in the diagnosis of ehrlichiosis, the importance of molecular methods is increasing steadily due to their high sensitivity and specificity. The aim of this study was to determine the analytical sensitivity of a conventional polymerase chain reaction (PCR) targeting Ehrlichia spp. disulfide oxidoreductase (DSB) gene. Ehrlichia spp. DSB gene was cloned into the TOPO vector. After TOPO plasmid containing DSB gene were serially diluted, PCR targeting the Ehrlichia spp. DSB gene was performed. While working on this research, blood and skin scraping samples of a stray dog clinically suspected with leishmaniasis as well as treated for leishmaniasis arrived to our laboratory. Thereafter, PCRs targeting Ehrlichia spp. DSB and 16S rRNA and Leishmania kinetoplast DNA (kDNA) genes were performed to identify the pathogen in blood and skin scraping samples of the stray dog. The analytical sensitivity of the PCR assay targeting Ehrlichia spp. DSB gene was 1 ≥ copy plasmid/reaction using serially diluted TOPO plasmid containing DSB gene. PCR targeting the Ehrlichia spp. DSB gene was positive and PCR targeting Leishmania spp. kDNA was negative in blood and skin samples of the stray dog clinically suspected with leishmaniasis. Using nested PCR targeting Ehrlichia spp. 16S rRNA, E. canis was identified in blood and skin scraping samples of the stray dog. In this study, PCR targeting Ehrlichia spp. DSB gene has been shown to have high sensitivity. Also it was shown molecular methods can help clinicians in differential diagnosis of ehrlichiosis and leishmaniasis to prevent inappropriate treatment.

Keywords

• Disulfide oxidoreductase
• Ehrlichia canis
• Ehrlichiosis
• PCR
• Leishmaniasis

Thanks

The authors would like to thank to Dr. Munir Aktaş and Dr. Sezayi Özübek from University of Fırat, Turkey for providing Ehrlichia positive DNA. Also, the authors would like to thank Esra Atalay for technical support.

References

1. 1. Paddock CD, Childs JE. Ehrlichia chaffeensis: A prototypical emerging pathogen. Clin Microbiol Rev. 2003;16(1):37–64.
2. 2. Hoşgör M, Bilgiç HB, Bakırcı S, Ünlü AH aka., Karagenç T, Eren H. Detection of Anaplasma / Ehrlichia Species of Cattle and Ticks in Aydın Region. Turkiye Parazitol Derg. 2015;39(4):291–8.
3. 3. Aysul N, Ural K, Cetinkaya H, Kuşkucu M, Toros G, Eren H, et al. Doxycycline-chloroquine combination for the treatment of canine monocytic ehrlichiosis. Acta Sci Vet. 2012;40(2).
4. 4. Maeda K, Markowitz N, Hawley R, Ristic M, Cox D, McDade J. Human infection with Ehrlichia canis, a leukocytic rickettsia. N Engl J Med. 1987;316(14):853–6.
5. 5. Unver A, Rikihisa Y, Kawahara M, Yamamoto S. Analysis of 16S rRNA gene sequences of Ehrlichia canis, Anaplasma platys, and Wolbachia species from canine blood in Japan. Ann N Y Acad Sci. 2003;990:692–8.
6. 6. Unver A, Rikihisa Y, Borku K, Ozkanlar Y, Hanedan B. Molecular detection and characterization of Ehrlichia canis from dogs in Turkey. Vol. 118, Berliner und Munchener Tierarztliche Wochenschrift. 2005. p. 300–4.
7. 7. Ristic M, Holland C. Canine ehrlichiosis. In: Rickettsial and chlamydial diseases of domestic animals. 1993. p. 169–86.
8. 8. Dodurka HT, Bakırel U. Bir Köpekte Ehrlichiosis Olgusu. İstanbul Üniversitesi Vet Fakültesi Derg. 2002;28(1):11–6.

9. 9. Aktas M, Özübek S, Altay K, Ipek NDS, Balkaya I, Utuk AE, et al. Molecular detection of tick-borne rickettsial and protozoan pathogens in domestic dogs from Turkey. Parasites and Vectors. 2015;8(1):4–9.
10. 10. Ciaramella P, Oliva G, De Luna R, Gradoni L, Ambrosio R, Cortese L, et al. A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by Leishmania infantum. Vet Rec. 1997;141(21):539–43.
11. 11. Atasoy A, Pasa S, Ozensoy Toz S, Ertabaklar H. Kıyı Ege Bölgesindeki Köpeklerde Visseral Leishmaniasis’in Seroprevalansı. Kafkas Univ Vet Fak Derg. 2009;16(1):1–6.
12. 12. Balcioǧlu IC, Ertabaklar H, Paşa S, Ozbel Y, Toz SO. Investigating the seroprevalance of leishmaniasis in four dog shelters in Antalya and its districts. Turkiye Parazitol Derg. 2009;33(1):4–7.
13. 13. Gültekin M, Paşa S, Ural K, Balıkçı C, Ekren Aşıcı GS, Gültekin G. Oxidative Status and Lipid Profile among Dogs at Different Stages of Visceral Leishmaniasis. Turkiye parazitolojii Derg. 2017;41(4):183–7.
14. 14. Bakirci S, Bilgiç HB, Köse O, Aksulu A, Hacilarlioğlu S, Erdoğan H, et al. Molecular and seroprevalence of canine visceral leishmaniasis in West Anatolia, Turkey. Turkish J Vet Anim Sci. 2016;40(5):637–44.
15. 15. Ansari-Mood M, Khoshnegah J, Mohri M, Rajaei SM. Seroprevalence and risk factors of ehrlichia canis infection among companion dogs of Mashhad, North East of Iran, 2009-2010. J Arthropod Borne Dis. 2015;9(2):184–94.
16. 16. Matjila PT, Leisewitz AL, Jongejan F, Penzhorn BL. Molecular detection of tick-borne protozoal and ehrlichial infections in domestic dogs in South Africa. Vet Parasitol. 2008;155(1–2):152–7.
17. 17. Harrus S, Waner T. Diagnosis of canine monocytotropic ehrlichiosis (Ehrlichia canis): An overview. Vet J. 2011;187(3):292–6.
18. 18. Nakaghi ACH, Machado RZ, Ferro JA, Labruna MB, Chryssafidis AL, André MR, et al. Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis. Rev Bras Parasitol Vet. 2010;19(2):1–5.
19. 19. Dantas-Torres F, Chomel BB, Otranto D. Ticks and tick-borne diseases: A One Health perspective. Trends Parasitol. 2012;28(10):437–46.
20. 20. Wen B, Rikihisa Y, Mott JM, Greene R, Kim HY, Zhi N, et al. Comparison of nested PCR with immunofluorescent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline. J Clin Microbiol. 1997;35(7):1852–5.
21. 21. Singu V, Peddireddi L, Sirigireddy KR, Cheng C, Munderloh U, Ganta RR. Unique macrophage and tick cell-specific protein expression from the p28/ p30-outer membrane protein multigene locus in Ehrlichia chaffeensis and Ehrlichia canis. Cell Microbiol. 2006;8(9):1475–87.
22. 22. Harrus S, Waner T, Aizenberg I, Foley JE, Poland AM, Bark H. Amplification of ehrlichial DNA from dogs 34 months after infection with Ehrlichia canis. J Clin Microbiol. 1998;36(1):73–6. 23. Seaman RL, Kania SA, Hegarty BC, Legendre AM, Breitschwerdt EB. Comparison of results for serologic testing and a polymerase chain reaction assay to determine the prevalence of stray dogs in eastern Tennessee seropositive to Ehrlichia canis. Am J Vet Res. 2004;65(9):1200–3.
23. 24. Sainz Á, Roura X, Miró G, Estrada-Peña A, Kohn B, Harrus S, et al. Guideline for veterinary practitioners on canine ehrlichiosis and anaplasmosis in Europe. Parasites and Vectors. 2015;8(1):1–20.
24. 25. Doyle CK, Labruna MB, Breitschwerdt EB, Tang YW, Corstvet RE, Hegarty BC, et al. Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene. J Mol Diagnostics. 2005;7(4):504–10.
25. 26. Aguiar DM, Hagiwara MK, Labruna MB. In vitro isolation and molecular characterization of an Ehrlichia canis strain from São Paulo, Brazil. Brazilian J Microbiol. 2008;39(3):489–93.
26. 27. Labruna MB, McBride JW, Camargo LMA, Aguiar DM, Yabsley MJ, Davidson WR, et al. A preliminary investigation of Ehrlichia species in ticks, humans, dogs, and capybaras from Brazil. Vet Parasitol. 2007;143(2):189–95.
27. 28. Döşkaya M, Caner A, Deǧirmenci A, Wengenack NL, Yolasiǧmaz A, Turgay N, et al. Degree and frequency of inhibition in a routine realtime PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey. J Med Microbiol. 2011;60(7):937–44.
28. 29. Can H, Inceboz T, Caner A, Atalay Şahar E, Karakavuk M, Döşkaya M, et al. Kist Örneklerinde Yeni Bir Tek Tüp Multipleks Gerçek Zamanli Polimeraz Zincir Reaksiyonu He Echinococcus granulosus ve Echinococcus multilocularis’ in Saptanmasi. Mikrobiyol Bul. 2016;50(2):266–77.
29. 30. Can H, Döşkaya M, Özdemir HG, Şahar EA, Karakavuk M, Pektaş B, et al. Seroprevalence of Leishmania infection and molecular detection of Leishmania tropica and Leishmania infantum in stray cats of I˙zmir, Turkey. Exp Parasitol. 2016;167:109–14.
30. 31. Anderson BE, Sumner JW, Dawson JE, Tzianabos T, Greene CR, Olson JG, et al. Detection of the etiologic agent of human ehrlichiosis by polymerase chain reaction. J Clin Microbiol. 1992;30(4):775–80.
31. 32. Dawson J, Biggie K, Warner C, Jenkins S, Levine J, Olson J. Polymerase chain reaction evidence of Ehrlichia chaffeensis, an etiologic agent of human ehrlichiosis, in dogs from southeast Virginia. Am J Vet Res. 1996;57(8):1175–9.
32. 33. Murphy GL, Ewing SA, Whitworth LC, Fox JC, Kocan AA. A molecular and serologic survey of Ehrlichia canis, E. chaffeensis, and E. ewingii in dogs and ticks from Oklahoma. Vet Parasitol. 1998;79(4):325–39.
33. 34. Noyes HA, Reyburn H, Bailey JW, Smith D. A nested-PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan. J Clin Microbiol. 1998;36(10):2877–81.
34. 35. Adao DE V., Herrera CMT, Galarion LH, Bolo NR, Carlos RS, Carlos ET, et al. Detection and molecular characterization of Hepatozoon canis, Babesia vogeli, Ehrlichia canis, and Anaplasma platys in dogs from Metro Manila, Philippines. Korean J Vet Res. 2017;57(2):79–88.
35. 36. Childs JE, Sumner JW, Nicholson WL, Massung RF, Standaert SM, Paddock CD. Outcome of diagnostic tests using samples from patients with culture- proven human monocytic ehrlichiosis: Implications for surveillance. J Clin Microbiol. 1999;37(9):2997–3000.
36. 37. Vieira RF da C, Biondo AW, Guimarães AMS, Santos AP dos, Santos RP dos, Dutra LH, et al. Ehrlichiosis in Brazil. Rev Bras Parasitol Veterinária. 2011;20(1):01–12.
37. 38. Paddock CD, Folk SM, Shore GM, Machado LJ, Huycke MM, Slater LN, et al. Infections with Ehrlichia chaffeensis and Ehrlichia ewingii in Persons Coinfected with Human Immunodeficiency Virus . Clin Infect Dis. 2001;33(9):1586–94.
38. 39. Standaert SM, Yu T, Scott MA, Childs JE, Paddock CD, Nicholson WL, et al. Primary Isolation of Ehrlichia chaffeensis from Patients with Febrile Illnesses: Clinical and Molecular Characteristics . J Infect Dis. 2000;181(3):1082–8.
39. 40. Ndip LM, Labruna M, Ndip RN, Walker DH, McBride JW. Molecular and clinical evidence of Ehrlichia chaffeensis infection in Cameroonian patients with undifferentiated febrile illness. Ann Trop Med Parasitol. 2009;103(8):719–25.
40. 41. Düzlü Ö, İnci A, Yıldırım A, Önder Z, Ciloğlu A. The investigation of vector-borne some protozoon and rickettsial infections in dogs by Real Time PCR and the molecular characterizations of the obtained isolates. Vet J Ankara Univ. 2014;61:275–82.
41. 42. Batmaz H, Nevo E, Waner T, Şentürk S, Yılmaz Z, Harrus S. Seroprevalence of Ehrlichia canis antibodies among dogs in Turkey. Vet Rec. 2001;148(21):665–6.
42. 43. Ozubek S, Sayın Ipek DN, Aktas M. A molecular survey of rickettsias in shelter dogs and distribution of rhipicephalus sanguineus (Acari: Ixodidae) sensu lato in Southeast Turkey. J Med Entomol. 2018;55(2):459–63.
43. 44. Çetinkaya H, Matur E, Akyazi İ, Ekiz EE, Aydin L, Toparlak M. Serological and molecular investigation of Ehrlichia spp. and Anaplasma spp. in ticks and blood of dogs, in the Thrace Region of Turkey. Ticks Tick Borne Dis. 2016;7(5):706–14.
44. 45. Silveira JAG, Valente PCLG, Paes PRO, Vasconcelos A V., Silvestre BT, Ribeiro MFB. The first clinical and laboratory evidence of co-infection by Anaplasma phagocytophilum and Ehrlichia canis in a Brazilian dog. Ticks Tick Borne Dis. 2015;6(3):242–5.