DESCRIPTION OF REVERSE LINE BLOTTING (RLB) TECHNIQUE AND RLB BASED STUDIES AIMED TO DETERMINE THEILERIA AND BABESIA SPECIES IN SHEEP AND GOATS IN TURKEY

Abstract

Ticks play a role in transmission of more than 200 pathogens of medical and veterinary importance. These pathogens are present in form of single or mixed infections in the vertebrate host and the vector ticks. Microscopic and serological methods have some disadvantages such as difficulties in species discrimination and cross-reaction problems. Elimination of these disadvantages has been possible by using molecular techniques. Recently reverse line blotting (RLB) method has been used for determination of multiple infections simultaneously. RLB is also useful molecular tool to discover new species and genotypes. RLB is consist of seven steps as the stages of genus level PCR, binding species-specific probes to the Biodyne-C membrane, denaturation of PCR products, hybridization of denatured products with probes, formation of streptavidin-POD + biotin complex, realization of the light emission of this complex and evaluation the result by transferring this on a radiographic film in a dark room. This review provides detailed information about the usage of RLB in the detection of tick-borne infections.

Keywords

• Tick,pathogen,PCR,RLB.

REVERSE LINE BLOTTING TEKNİĞİNİN AÇIKLANMASI VE TÜRKİYE’DE KOYUN VE KEÇİLERDE THEİLERİA VE BABESİA TÜRLERİNİN BELİRLENMESİNE YÖNELİK RLB TABANLI ÇALIŞMALAR

Abstract

Keneler tıbbi ve veteriner öneme sahip 200’den fazla patojenin naklinde rol oynamaktadırlar. Bu etkenler omurgalı konakta ve vektör kenelerde tek veya miks enfeksiyonlar şeklinde bulunmaktadır. Mikroskobik ve serolojik yöntemler tür ayrımında karşılaşılan zorluklar ve çapraz reaksiyon gibi dezavantajlara sahiptir. Bu dezavantajların giderilmesi moleküler tanı metotlarının kullanılmaya başlanması ile mümkün olabilmektedir. Son yıllarda, Reverse Line Blotting (RLB) metodu kullanılarak çoklu enfeksiyonların aynı zaman diliminde tespiti yapılabilmektedir. RLB metodu, yeni tür ve genotiplerin keşfedilmesini sağlayan moleküler bir tanı metodudur. RLB testi; cins düzeyinde PZR, türe özgü probların Biodyne-C membrana bağlanması, PZR ürünlerinin denatürasyonu, denatüre ürünlerin problar ile hibridizasyonu, streptavidin-POD + biotin kompleksinin oluşumu, bu kompleksin ışık emisyonunun gerçekleşmesi ve bunun karanlık odada radyografik film üzerine aktarılarak değerlendirilmesi olmak üzere yedi aşamadan oluşmaktadır. Bu derlemede kenelerle bulaşan hastalıkların teşhisinde RLB metodunun kullanımı hakkında detaylı bilgi verilmektedir.

Keywords

• Kene,Patojen,PZR,RLB

References

1. 1. Friedhoff KT. Tick-borne disease of sheep and goats caused by Babesia, Theileria or Anaplasma spp. Parassitologia. 1997;39:99-109.
2. 2. Jongejan F, Uilenberg G. The global importance of ticks. Parasitology. 2004;129(Suppl):3-14.
3. 3. Guo S, Yuan Z, Wu G, et al. Epidemiology of ovine theileriosis in Ganan region, Gansu Province, China. Parasitol Res. 2002;88:36-7.
4. 4. Yin H, Liu G, Luo J, et al. Observation on the schizont stage of an unidentified Theileria sp. in experimentally infected sheep. Parasitol Res. 2003;91(1):34-9.
5. 5. Brown CG. Control of tropical theileriosis (Theileria annulata infection) of cattle. Parassitologia. 1990;32(1):23-31.
6. 6. Uilenberg G: Theilerial Species of Domestic Livestock. “ Irvın AD, Cunningham MP, Young AS. (eds): Advences in the Control of Theileriosis, 2nd ed” pp.4-37, Martinus Nijhoff Publishers, London (1981).
7. 7. Burridge MJ, Brown CG, Kimber CD. Theileria annulata: cross-reactions between a cell culture schizont antigen and antigens of East African species in the indirect fluorescent antibody test. Exp Parasitol. 1974;35(3):374-80.
8. 8. Gubbels MJ, D’olıveıra C, Jongejan F. Development of an indirect Tamsl enzyme-linked immunosorbent assay for diagnosis of Theileria annulata infection in cattle. Clin Diagn Lab Immunol. 2000;7:404-11.

9. 9. Leemans I, Brown D, Hooshmand-Rad P, Kirvar E, Uggla A. Infectivity and cross-immunity studies of Theileria lestoquardi and Theileria annulata in sheep and cattle: I. In vivo responses. Vet Parasitol. 1999;82(3):179-92.
10. 10. Aktas M, Altay K, Dumanli N. Determination of prevalence and risk factors for infection with Babesia ovis in small ruminants from Turkey by polymerase chain reaction. Parasitol Res. 2007;100(4):797-802.
11. 11. Altay K, Aktas M, Dumanli N, Aydin MF. Evaluation of a PCR and comparison with RLB for detection and differentiation of Theileria sp. MK and other Theileria and Babesia species of small ruminants. Parasitol Res. 2008a;103(2):319-23.
12. 12. Altay K, Aydin MF, Dumanli N, Aktas M. Molecular detection of Theileria and Babesia infections in cattle. Vet Parasitol. 2008b;158(4):295-301.
13. 13. Altay K, Aydin MF, Uluisik U, Aktas M, Dumanli N. Use of multiplex PCR for the diagnosis of Theileria annulata and Theileria buffeli. Turkiye Parazitol Derg. 2008c;32(1):1-3.
14. 14. D'oliveira C, Van Der Weide M, Habela MA, Jacquiet P, Jongejan F. Detection of Theileria annulata in blood samples of carrier cattle by PCR. J Clin Microbiol. 1995;33:2665-9.
15. 15. Gubbels JM, De Vos AP, Van Der Weide M, et al. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. J Clin Microbiol. 1999;37:1782-9.
16. 16. Nagore D, García-Sanmartín J, García-Pérez AL, Juste RA, Hurtado A. Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Northern Spain. Int J Parasitol. 2004;34:1059-67.
17. 17. Schnittger L, Yin H, Qi B, et al. Simultaneously detection and differentiation of Theileria and Babesia parasite infecting small ruminants by reverse line blotting. Parasitol Res. 2004;92:189-96.
18. 18. Vatansever Z, Iça A, Deniz A, et al. Ankara Yöresinde kene kaynaklı protozoon enfeksiyonlarının reverse line blotting (RLB) ve indirek floresan antikor testi (IFAT) ile saptanması. XIII. Ulusal Parazitoloji Kongresi. Konya: Selcuk University, 2003, p: 94.
19. 19. Altay K, Dumanli N, Aktas M. Molecular identification, genetic diversity and distribution of Theileria and Babesia species infecting small ruminants. Vet Parasitol. 2007b;147:161-5.
20. 20. Guan G, Moreau E, Liu J, et al. Babesia sp. BQ1 (Lintan): Molecular evidence of experimental transmission to sheep by Haemaphysalis qinghaiensis and Haemaphysalis longicornis. Parasitol Int. 2010;59(2):265-7.
21. 21. Guan GQ, Ma ML, Moreau E, et al. A new ovine Babesia species transmitted by Hyalomma anatolicum anatolicum. Exp Parasitol. 2009;122:261-7.
22. 22. Liu AH, Yin H, Guan GQ, et al. At least two genetically distinct large Babesia species infective to sheep and goats in China. Vet Parasitol. 2007;147:246-51.
23. 23. Niu Q, Luo J, Guan G, et al. Detection and differentiation of ovine Theileria and Babesia by reverse line blotting in China. Parasitol Res. 2009;104(6):1417-23.
24. 24. Rijpkema SG, Molkenboer MJ, Schouls LM, Jongejan F, Schellekens JF. Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes. J Clin Microbiol. 1995;33:3091-5.
25. 25. Sarayli H, Inci A, Ica A, Yildirim A, Duzlu O. Yeşilhisar yöresindeki koyun ve keçilerde Babesia etkenlerinin Reverse Line Blotting (RLB) yöntemiyle araştırılması. Erciyes Universitesi Sağlık Bilimleri Dergisi. 2006;15(3):181-8.
26. 26. Iça A, Yildirim A, Inci A. Kayseri yöresinde koyunlarda kan protozoonlarının Reverse Line Blotting Yöntemi ile Araştırılması. XIV Ulusal Parazitoloji Kongresi, 2005, İzmir: Ege University, p: 161.
27. 27. Aktas M, Altay K, Dumanli N. Survey of Theileria parasites of sheep in eastern Turkey using polymerase chain reaction. Small Rum Res. 2005b;60:289-93.
28. 28. Altay K, Aktas M, Dumanli N. Theileria infections in small ruminants in the east and southeast Anatolia. Turkiye Parazitol Derg. 2007a;31(4):268-71.
29. 29. Inci A, Ica A, Yildirim A, Duzlu O. Identification of Babesia and Theileria species in small ruminants in Central Anatolia (Turkey) via reverse line blotting. Turk J Vet Anim Sci. 2010;34(2):205-10.
30. 30. Aydin MF, Aktas M, Dumanli N. Molecular identification of Theileria and Babesia in sheep and goats in the Black Sea Region in Turkey. Parasitol Res. 2013;112(8):2817-24.
31. 31. Aktas M, Altay K, Dumanli N. Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats. Vet Parasitol. 2005a;133:277-81.
32. 32. Georges K, Loria GR, Riili S, et al. Detection of haemoparasites in cattle by reverse line blot hybridisation with a note on the distribution of ticks in Sicily. Vet Parasitol. 2001;99(4):273-86.
33. 33. Anonymous. Principles of the Reverse Line Blot Hybridization Assay. http://www.icttd.nl/fileadmin/user_upload/Presentations/Nijhof001.zip [accessed 22 November 2012].