Molecular detection and genotyping of Theileria equi infection within the equine population in Giza, Egypt, using real-time PCR as compared with conventional detection methods

Document Type : Full paper (Original article)

Authors

1 Biotechnology Department, Animal Health Research Institute, Agricultural Research Center, Dokki 12618, Giza, Egypt

2 Parasitology Department, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516 Kafrelsheikh, Egypt

3 Graduated from Faculty of Medicine, Mansoura University, 35516 Mansoura, Egypt

4 Department of Poultry Husbandry, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tando Jam 70060, Pakistan

5 Ph.D. Student in Infectious Diseases, Transboundary Animal Diseases Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Korimoto, Kagoshima 8900065, Japan

6 Parasitology Department, Faculty of Veterinary Medicine, Minia University, Minya, Egypt

10.22099/ijvr.2025.51028.7553

Abstract

Background: Equine piroplasmosis represents one of the main and serious health problems affecting the equines industry globally, caused by a tick-borne protozoa called Theileria equi and Babesia caballi. Aims: This study aimed to identify and genotype T. equi within the equine population from Giza Governorate, Egypt, by comparing the obtained results using the available diagnostic methods. Methods: We collected 116 apparently healthy horses from the study area during the first half of 2019 to identify T. equi using real-time PCR (qPCR), targeting the 18S rRNA gene. The results were compared with those from microscopic examination of Giemsa-stained blood smears and conventional PCR. Genotyping of the obtained sequences was also conducted to explore the genetic diversity of the detected T. equi strains. Results: Through sequencing and phylogenetic analysis, our samples were grouped into clusters corresponding to genotype A and genotype E. Our results demonstrated that the qPCR had the highest sensitivity (100%) followed by conventional PCR (68%) while microscopic examination had the lowest sensitivity (38%). Furthermore, the negative predictive value (NPV) of qPCR was the highest (100%) compared with conventional PCR and microscopical examination (80.49% and 68.04%, respectively) which revealed that negative cases detected by qPCR were certainly correct compared with the other two diagnostic assays. Conclusion: It is highly recommended to incorporate PCR diagnostic assays alongside microscopic examination to evaluate the epidemiological status of equine piroplasmosis. Also, our study demonstrated that T. equi genotype A and genotype E are circulating among Egyptian horses.

Keywords

Main Subjects

References

Abdel-Shafy, S; Abdullah, HHAM; Elbayoumy, MK; Elsawy, BSM; Hassan, MR; Mahmoud, MS; Hegazi, AG and AbdelRahman, EH (2022). Molecular epidemiological investigation of piroplasms and anaplasmataceae bacteria in Egyptian domestic animals and associated ticks. Pathogens. 11: 1194.
Abdullah, HAM; Aboelsoued, D; Farag, TK; Abdel-Shafy, S; Abdel Megeed, KN; Parola, P; Raoult, D and Mediannikov, O (2022). Molecular characterization of some equine vector-borne diseases and associated arthropods in Egypt. Acta Trop., 227: 106274. https://doi. org/10.1016/j.actatropica.2021.106274.
Abedi, V; Razmi, G; Seifi, H and Naghibi, A (2015). Molecular detection of equine piroplasms in donkeys (Equus asinus) in North Khorasan province, Iran. Iran. J. Vet. Res., 16: 202-204.
Ahedor, B; Otgonsuren, D; Zhyldyz, A; Guswanto, A; Ngigi, NMM; Valinotti, MFR; Kothalawala, H; Kalaichelvan, N; Silva, SSP; Kothalawala, H; Acosta, TJ; Sivakumar, T and Yokoyama, N (2023a). Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes. Parasit. Vectors. 16: 435-444. doi: 10.1186/s13071-023-06045-z.
Ahedor, B; Sivakumar, T; Valinotti, MFR; Otgonsuren, D; Yokoyama, N and Acosta, JT (2023b). PCR detection of Theileria equi and Babesia caballi in apparently healthy horses in Paraguay. Vet. Parasitol. Reg. Stud. Rep., 39: 100835. https://doi.org/10.1016/j.vprsr.2023.100835.
Alanazi, AD; Said, AE; Morin-Adeline, V; Alyousif, MS and Šlapeta, J (2014). Quantitative PCR detection of Theileria equi using laboratory workflows to detect asymptomatic persistently infected horses. Vet. Parasitol., 206: 138-145.
Alhassan, A; Pumidonming, W; Okamura, M; Hirata, H; Battsetseg, B; Fujisaki, K; Yokoyama, N and Igarashi, I (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.
Altay, K; Erol, U; Sahin, OF; Ulucesme, MC; Aytmirzakizi, A and Aktas, M (2024). Survey of tick-borne pathogens in grazing horses in Kyrgyzstan: phylogenetic analysis, genetic diversity, and prevalence of Theileria equi. Front. Vet. Sci., 11: 1359974. doi: 10.3389/fvets.2024.1359974.
Amer, MM; Galon, EM; Soliman, AM; Do, T; Zafar, I; Ma, Y; Li, H; Ji, S; Mohanta, UK and Xuan, X (2024). Molecular detection of tick-borne piroplasmids in camel blood samples collected from Cairo and Giza governorates, Egypt. Acta Trop., 256: 107252. doi: 10.1016/j.actatropica. 2024.107252.
Ana, MV; Ana, PDSDL; Guilherme, BW; Rodrigo, CC and Fábio, PLL (2018). Equine theileriosis: Review. Ann. Rev. Resear., 3: 555620.
Asif, M; Parveen, A; Ashraf, S; Hussain, M; Aktas, M; Ozubek, S; Shaikh, RS and Iqbal, F (2020). First report regarding the simultaneous molecular detection of Anaplasma marginale and Theileria annulata in equine blood samples collected from Southern Punjab in Pakistan. Acta Parasit., 65: 259-263. https://doi.org/10.2478/s11686-019-00141-w.
Bhagwan, J; Kumar, A; Kumar, R; Goyal, L; Goel, P and Kumar, S (2015). Molecular evidence of Theileria equi infection in Hyalomma anatolicum ticks infested on sero-positive Indian horses. Acta Parasitol., 60: 322-329.
Bhoora, RV; Collins, NE; Schnittger, L; Troskie, C; Marumo, R; Labuschagne, K; Smith, RM; Dalton, DL and Mbizeni, S (2020). Molecular genotyping and epidemiology of equine piroplasmids in South Africa. Ticks Tick-Borne Dis., 11: 101358.
Bhoora, R; Franssen, L; Oosthuizen, MC; Guthrie, AJ; Zweygarth, E; Penzhorn, BL; Jongejan, F and Collins, NE (2009). Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa. Vet. Parasitol., 159: 112-120.
Bhoora, R; Quan, M; Franssen, L; Butler, CM; Van der Kolk, JH; Guthrie, AJ; Zweygarth, E; Jongejan, F and Collins, NE (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.
Ceylan, O; Benedicto, B; Ceylan, C; Tumwebaze, M; Galon, EM; Liu, M; Xuan, X and Sevinc, F (2021). A survey on equine tick-borne diseases: The molecular detection of Babesia ovis DNA in Turkish racehorses. Ticks Tick Borne Dis., 12: 101784. doi: 10.1016/j.ttbdis. 2021.101784.
Davitkov, D; Vucicevic, M; Stevanovic, J; Krstic, V; Slijepcevic, D; Glavinic, U and Stanimirovic, Z (2016). Molecular detection and prevalence of Theileria equi and Babesia caballi in horses of central Balkan. Acta Parasitol., 61: 337-342.
Elsawy, BSM; Nassar, AM; Alzan, HF; Bhoora, RV; Ozubek, S; Mahmoud, MS; Kandil, OM and Mahdy, OA (2021). Rapid detection of equine piroplasms using multiplex PCR and first genetic characterization of Theileria haneyi in Egypt. Pathogens. 10: 1414.
Elseify, MA; Helmy, NM; Elhawary, NM and Soliman, AM (2018). Using molecular techniques as an alternative tool for diagnosis and characterization of Theileria equi. IJVS., 32: 5-11.
Farah, AW; Hegazy, NA; Romany, MM and Soliman, YA (2003). Molecular detection of Babesia equi in infected and carrier horses by polymerase chain reaction. EJI. 10: 73-79.
Francisco, MJ; Alejandro, PÉ; Lowell, KS; Carlos, SE and Reginaldo, BG (2024). New insights in the diagnosis and treatment of equine piroplasmosis: pitfalls, idiosyncrasies, and myths. Front. Vet. Sci., 11: 1459989. doi: 10.3389/ fvets.2024.1459989.
Ibrahim, AK; Gamil, IS; Abd-El baky, AA; Hussein, MM and Tohamy, AA (2011). Comparative molecular and conventional detection methods of Babesia equi (B. Equi) in Egyptian equine. Glob. Vet., 7: 201-210.
Jaffer, O; Abdishakur, F; Hakimuddin, F; Riya, A; Wernery, U and Schuster, RK (2009). A comparative study of serological tests and PCR for the diagnosis of equine piroplasmosis. Parasitol. Res., 106: 709-713.
Kim, C; Blanco, LBC; Alhassan, A; Iseki, H; Yokoyama, N; Xuan, X and Igarashi, I (2008). Diagnostic real-time PCR assay for the quantitative detection of Theileria equi from equine blood samples. Vet. Parasitol., 151: 158-163.
Knowles, DP; Kappmeyer, LS; Haney, D; Herndon, DR; Fry, LM; Munro, JB; Sears, K; Ueti, MW; Wise, LN and Silva, M (2018). Discovery of a novel species, Theileria haneyi n. sp. infective to equids, highlights exceptional genomic diversity within the genus Theileria: Implications for apicomplexan parasite surveillance. Int. J. Parasitol., 48: 679-690.
Kumar, S; Stecher, G; Li, M; Knyaz, C and Tamura, K (2018). MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol., 35: 1547-1549. https://doi.org/10.1093/molbev/msy096.
Kumar, S; Sudan, V; Shanker, D and Devi, A (2020). Babesia (Theileria) equi genotype A among Indian equine population. Vet. Parasitol. Reg. Stud. Rep., 19: 100367. https://doi.org/10.1016/j.vprsr.2019.100367.
Maharana, BR; Ganguly, A; Potliya, S; Kumar, B; Singh, H; Dash, A and Khanna, S (2024). Molecular detection and characterization of prevailing Theileria equi genotype in equine from Northern India. Res. Vet. Sci., 173: 105277. https://doi.org/10.1016/j.rvsc.2024.105277.
Mahdy, OA; Nassar, AM; Mohamed, BS and Mahmoud, MS (2016). Comparative diagnosis utilizing molecular and serological techniques of Theileria equi infection in distinct equine populations in Egypt. Int. J. Chem. Tech. Res., 9: 185-197.
Mahmoud, MS; El-Ezz, NTA; Abdel-Shafy, S; Nassar, SA; Namaky, AH; Khalil, WKB; Knowles, D; Kappmeyer, L; Silva, MG and Suarez, CE (2016). Assessment of Theileria equi and Babesia caballi infections in equine populations in Egypt by molecular, serological and hematological approaches. Parasit. Vectors. 9: 1-10.
Mahmoud, MS; Kandil, OM; Abu Ezz, NTE; Hendawy, SHM; Elsawy, BSM; Knowles, DP; Bastos, RG; Kappmeyer, LS; Laughery, JM and Alzan, HF (2020). Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4). Parasit. Vectors. 13: 369-378.
Malekifard, F; Tavassoli, M; Yakhchali, M and Darvishzadeh, R (2014). Detection of Theileria equi and Babesia caballi using microscopic and molecular methods in horses in suburb of Urmia, Iran. Vet. Res. Forum. 5: 129-133.
Otgonsuren, V; Amgalanbaatar, T; Narantsatsral, S; Enkhtaivan, B; Munkhgerel, D; Zoljargal, M;
Davkharbayar, B; Myagmarsuren, P; Battur, B; Battsetseg, B; Sivakumar, T and Yokoyama, N (2024). Epidemiology and genetic diversity of Theileria equi and Babesia caballi in Mongolian horses. Inf. Gen. Evol., 119: 105571. https://doi.org/10.1016/j.meegid.2024.105571.
Ozubek, S and Aktas, M (2018). Genetic diversity and prevalence of piroplasm species in equids from Turkey. Comp. Immunol. Microbiol. Infect. Dis., 59: 47-51.
Radwan, AMM (2009). An investigation on parasitic infection in equines. Thesis, Parasitology Department, Faculty of Veterinary Medicine, Banha University.
Ramadan, RM; Taha, NM; Auda, HM; Elsamman, EM; El-Bahy, MM and Salem, MA (2024). Molecular and immunological studies on Theileria equi and its vector in Egypt. Exp. Appl. Acarol., 93: 439-458. https://doi.org/10. 1007/s10493-024-00933-4.
Raza, A; Ijaz, M; Mehmood, K; Ahmed, A; Javed, MU; Anwaar, F; Rasheed, H and Ghumman, NZ (2024). Theileria equi infection in working horses of Pakistan: Epidemiology, molecular characterization, and hemato-biochemical analysis. J. Parasitol., 110: 79-89.
Rothschild, CM (2013). Equine piroplasmosis. J. Equine Vet. Sci., 33: 497-508.
Sadeddine, R; Righi, S; Saidani, K and Benakhla, A (2025). First molecular characterization of Theileria equi from Northeastern Algeria. Acta Parasit., 70: 66. https://doi.org/ 10.1007/s11686-025-01006-1.
Salib, FA; Youssef, RR; Rizk, LG and Said, SF (2013). Epidemiology, diagnosis and therapy of Theileria equi infection in Giza, Egypt. Vet. World. 6: 76-82.
Souza, EAR; Araujo, AC; Pires, L; Freschi, CR; Azevedo, SS; Machado, RZ and Horta, MC (2019). Serological detection and risk factors for equine piroplasmosis in the semiarid region of Pernambuco, Northeastern Brazil. Rev. Bras. Parasitol. Vet., 28: 685-691.
Sumbria, D; Singla, L; Amrita, S and Kaur, P (2015). Detection of Theileria species infecting equine population in Punjab by 18S rRNA PCR. Indian J. Anim. Res., 50: 218-223.
Thrusfield, M and Christley, R (2018). Veterinary epidemiology. 4th Edn., Wiley-Blackwell.
Zhang, Y; Shi, O; Laven, R; Li, C; He, W; Zheng, H; Liu, S; Lu, M; Yang, D; Guo, O and Chahan, B (2023). Prevalence and genetic diversity of Theileria equi from horses in Xinjiang Uygur Autonomous region, China. Ticks Tick-borne Dis., 14: 102193. https://doi.org/10.1016/j. ttbdis.2023.102193.

Files

Share

How to cite

Statistics