Molecular detection and phylogenetic analysis of Theileria equi and Babesia caballi in wild horses in Konya province of Turkey

Abstract

The aim of this study was to investigate equine piroplasms of wild horses (Equus ferus caballus) in Konya province of Turkey in November-December 2017. For this aim, blood samples were collected from 36 wild horses and examined for equine piroplasms by microscopy and multiplex PCR. Some of the PCR products from positive samples were also sequenced. Five (13.89%) out of the 36 horses were infected with either Theileria equi, Babesia caballi or both in the microscopical examination. Single infections with T. equi and B. caballi were detected in three (8.33%) and one horses (2.78%), respectively. Prevalence of T. equi, B. caballi and mix infections was determined as 50%, 38.8% and 38.8% by multiplex PCR, respectively. Multiplex PCR was found more sensitive than microscopical examination to detect the piroplasms of horses. The results of sequence analysis showed 99.25-100% and 98.23-99.59% nucleotide sequence identity to the previously reported T. equi and B. caballi 18S rRNA gene sequences, respectively. Consequently, the existence of equine piroplasmosis in wild horses was reported for the first time in Turkey, and high molecular prevalences of T. equi and B. caballi were reported with this study.

Keywords

• Babesia caballi
• multiplex PCR
• Theileria equi
• Turkey
• wild horse

References

1. Acici M, Umur S, Guvenc T, et al (2008): Seroprevalence of equine babesiosis in the Black Sea region of Turkey. Parasitol Int, 57, 198-200.
2. Akkan HA, Karaca M, Tutuncu M, et al (2003): Serologic and microscopic studies on babesiosis in horses in the Eastern border of Turkey. J Equine Vet Sci, 23, 181–183.
3. Aksoy AR (2016): At yetiştiriciliği ders notları. Available at https://docplayer.biz.tr/22854819-At-yetistiriciligi-ders-notlari-yrd-doc-dr-ali-riza-aksoy-1993-guncelleme-mayis-2016.html. (Accessed March 26, 2020).
4. Alhassan A, Pumidonming W, Okamura M, et al (2005): Development of a single round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood. Vet Parasitol, 129, 43–49.
5. Allsopp MT, Allsopp BA (2006): Molecular sequence evidence for the reclassification of some Babesia species. Ann N Y Acad Sci, 1081, 509–517.
6. Allsopp MTEP, Lewis BD, Penzhorn BL (2007): Molecular evidence for transplacental transmission of Theileria equi from carrier mares to their apparently healthy foals. Vet Parasitol, 148, 130–136.
7. Balkaya I, Erdogmus Z (2006): Investigation of prevalence of Babesia equi (Laveran, 1901) and Babesia caballi (Nuttall 1910) in horses by serological methods in Elazığ and Malatya province. Fırat Üniv Sag Bil Derg, 20, 61–63.
8. Baptista AP, Franco C, Mendes M, et al (2010): Subunit composition of Rhodothermus marinus respiratory complex I. Anal Biochem, 407, 104–110.

9. Baptista C, Lopes MS, Tavares AC, et al (2013): Diagnosis of Theileria equi infections in horses in the Azores using cELISA and nested PCR. Ticks Tick Borne Dis, 4, 242–245.
10. Bashiruddin JB, Cammà C, Rebêlo E (1999): Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16s rRNA gene. Vet Parasitol, 84, 75–83.
11. Bhoora R (2009): Molecular characterization of Babesia caballi and Theileria equi, the aetiological agents of equine piroplasmosis, in South Africa. PhD. Thesis. University of Pretoria, South Africa.
12. Bhoora R, Quan M, Franssen L, et al (2010): Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa. Vet Parasitol, 168, 201-211.
13. Boldbaatar D, Xuan X, Battsetseg B, et al (2005): Epidemiological study of equine piroplasmosis in Mongolia. Vet Parasitol, 127, 29-32.
14. Bose R, Jorgensen WK, Dalgliesh RJ, et al (1995): Current state and future trends in the diagnosis of babesiosis. Vet Parasitol, 57, 61-74.
15. Bruning A (1996): Equine piroplasmosis: an update on diagnosis, treatment and prevention. Br Vet J, 152, 139-151.
16. Bruning A, Phipps P, Posnett E, et al (1997): Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis. Vet Parasitol, 58, 11-26.
17. Calder JA, Reddy GR, Chieves LP, et al (1996): Monitoring Babesia bovis infections in cattle by using PCRbased tests. J Clin Microbiol, 11, 2748-2755.
18. De Waal DT (1992): Equine piroplasmosis: A review. Br Vet J, 148, 6-14.
19. Deniz A, Karaer Z, Cakmak A, et al (2007): Türkiye’de atlarda Babesia etkenlerinin saptanmasında PCR tekniğinin uygulanması (TAGEM, Project number: HS/01/02/06/05/103).
20. Edwards RZ, Moore H, LeRoy BE, et al (2011): Equine Babesiosis. Available at http://www.vet.uga.edu/vpp/clerk/edwards/index.php. (Accessed October 15, 2019).
21. Fahrimal Y, Gott WL, Jamer DP (1992): Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA. J Clin Microbiol, 30, 1374-1379.
22. Figueroa JV, Chieves LP, Johnson GS, et al (1993): Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis, and Anaplasma marginale DNA in bovine blood. Vet Parasitol, 50, 69-81.
23. Friedhoff KT (1988): Transmission of Babesia. 23-52. In: M Ristic (Ed), Babesiosis of Domestic Animals and Man. CRC Press, Boca Raton FL.
24. Guclu HZ, Karaer Z (2007): Detection of Babesia caballi (Nuttall, 1910) and Theileria equi (Syn. Babesia equi, Laveran, 1901) by the Polymerase Chain Reaction (PCR) in show and sport horses in the region of Ankara. Türkiye Parazitol Derg, 31, 89-93.
25. Guven E, Avcioglu H, Deniz A, et al (2017): Prevalence and molecular characterization of Theileria equi and Babesia caballi in jereed horses in Erzurum, Turkey. Acta Parasitol, 62, 207-213.
26. Heim A, Passos LMF, Ribeiro MFB, et al (2007): Detection and molecular characterization of Babesia caballi and Theileria equi isolates from endemic areas of Brazil. Parasitol Res, 102, 63-68.
27. Hirata H, Xuan X, Yokoyama N, et al (2003): Identification of a specific antigenic region of the P82 protein of Babesia equi and its potential use in serodiagnosis. J Clin Microbiol, 41, 547-551.
28. Hodgson J (2002): Equine exotic diseases, a manual for horse owners. Union Offset, Canberra.
29. Ikadai H, Osorio CR, Xuan X, et al (2000): Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein. Int J Parasitol, 30, 633-635.
30. Inci A (1997): Detection of Babesia caballi (Nuttall, 1910) and Babesia equi (Laveran, 1901) in horses by microscopic examination in military farm in Gemlik. Turk J Vet Anim Sci, 21, 43-46.
31. Inci A (2002): Investigation of the prevalence of Babesia equi (Laveran, 1901) and Babesia caballi (Nuttall, 1910) in equids by microscopic examination in Kayseri region. Fırat Üniv Sag Bil Derg, 1, 85-88.
32. Kappmeyer LS, Perryman LE, Hines SA, et al (1999): Detection of equine antibodies to Babesia caballi by recombinant B. caballi rhoptry-associated protein 1 in a competitive-inhibition enzyme-linked immunosorbent assay. J Clin Microbiol, 37, 2285-2290.
33. Karaman Il Cevre ve Orman Mudurlugu (2010): Karadağ mevkiindeki Yılkı atlarının durum tespiti, genel sağlık durumları, üremelerinin kontrolü ve çevre üzerindeki etkilerinin değerlendirilmesi projesi. Mevlana Kalkınma Ajansı Teknik Destek Projesi, Referans No: TR52-10-TD01.
34. Kerber CE, Ferreira F, Pereira MC (1999): Control of equine piroplasmosis in Brazil. Onderstepoort J Vet Res, 66, 123-127.
35. Kizilarslan F, Yildirim A, Duzlu O, et al (2015): Molecular detection and characterization of Theileria equi and Babesia caballi in horses (Equus ferus caballus) in Turkey. J Equine Vet Sci, 35, 830-835.
36. Krause PJ (2003): Babesiosis diagnosis and treatment. Vector Borne Zoonotic Dis, 3, 45-51.
37. Kumar S, Kumar Y, Malhotra DV, et al (2003): Standardization and comparison of serial dilution and single dilution elisa using different antigenic preparations of the Babesia equi parasite. Vet Res, 34, 71-83.
38. Kurt C, Yaman M (2005): The investigation of the prevalence Babesia equi and Babesia caballi in horses by microscopic and serologic (cELISA) methods in Adana province. YYU Vet Fak Derg, 23, 1-4.
39. Moretti A, Mangili V, Salvatori R, et al (2010): Prevalence and diagnosis of Babesia and Theileria infections in horses in Italy: A preliminary study. Vet J, 184, 346-350.
40. Nicolaiewsky TB, Richter MF, Lunge VR, et al (2001): Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction. Vet Parasitol, 101, 9-21.
41. Oncel T, Vural G, Gicik Y, et al (2007): Detection of Babesia (Theileria) equi (Laveran 1901) in Horses in the Kars Province of Turkey. Türkiye Parazitol Derg, 31, 170-172.
42. Ozubek S, Aktas M (2018): Genetic diversity and prevalence of piroplasm species in equids from Turkey. Comp Immunol Microbiol Infect Dis, 59, 47-51.
43. Quintao-Silva MG, Ribeiro MF (2003): Infection rate of Babesia spp. sporokinetes in engorged Boophilus microplus from an area of enzootic stability in the State of Minas Gerais, Brazil. Mem Inst Oswaldo Cruz, 98, 999-1002.
44. Rampersad J, Cesar E, Campbell MD (2003): A field evaluation of PCR for the routine detection of Babesia equi in horses. Vet Parasitol, 114, 81-87.
45. Saal JR (1964): Giemsa stain for the diagnosis of bovine babesiosis. II. Changes in erythrocytes infected with Babesia bigemina and B. argentina. J Protozool, 11, 582-585.
46. Sari B, Kirmizigul AH, Deniz A, et al (2009): Seroprevalence of Babesia caballi and Theileria equi in horses from Kars and Ardahan provinces during the winter season. Kafkas Üniv Vet Fak Derg, 16, 657-661.
47. Seifi HA, Mohri M, Sardari KA (2000): Mixed Infection of Babesia equi and Babesia caballi in a racing colt: a report from Iran. J Equine Vet Sci, 20, 858-860.
48. Sloboda M, Jirků M, Lukešová D, et al (2011): A survey of piroplasmids in horses and Bactrian camels in North-Eastern Mongolia. Vet Parasitol, 179, 246-249.
49. Tamzali Y (2013): Equine piroplasmosis: an updated review. Equine Vet Educ, 25, 590-598.
50. Uilenberg G (2006): Babesia-A historical overview. Vet Parasitol, 138, 3-10.
51. Wise LN, Kappmeyer LS, Mealey RH, et al (2013): Review of equine piroplasmosis. J Vet Intern Med, 27, 1334-1346.