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Veterinary parasitology2005; 129(1-2); 43-49; doi: 10.1016/j.vetpar.2004.12.018

Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood.

Abstract: With the aim of developing more simple diagnostic alternatives, a differential single-round and multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of Babesia caballi and Babesia equi, by targeting 18S ribosomal RNA genes. The multiplex PCR amplified DNA fragments of 540 and 392 bp from B. caballi and B. equi, respectively, in one reaction. The PCR method evaluated on 39 blood samples collected from domestic horses in Mongolia yielded similar results to those obtained from confirmative PCR methods that had been established earlier. Thus, the single-round and multiplex PCR method offers a simple tool for the differential diagnosis of B. caballi and B. equi infections in routine diagnostic laboratory settings as well as in epidemiological studies.
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Publication Date: 2005-04-09 PubMed ID: 15817201DOI: 10.1016/j.vetpar.2004.12.018Google Scholar: Lookup
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research paper describes a single-round and multiplex Polymerase Chain Reaction (PCR) method for the dual detection of Babesia caballi and Babesia equi in horse blood. This method aims to simplify the diagnostic process by targeting the 18S ribosomal RNA genes of the diseases.

Objective of the Research

  • The primary aim of this study was to develop an easier alternative for diagnostic processes by creating a differential single-round and multiplex PCR method. The method was designed to simultaneously detect Babesia caballi and Babesia equi by targeting 18S ribosomal RNA genes.

Material and Method

  • The multiplex PCR method, in the study, specifically amplified DNA fragments of 540 and 392 base pairs (bp) from Babesia caballi and Babesia equi respectively, in a single reaction. The ability to amplify specific DNA fragments from both parasites simultaneously is the key advantage of the process.

Results and Evaluation

  • This PCR method was evaluated on 39 blood samples collected from domestic horses in Mongolia. The results from this single-round and multiplex PCR method mirrored those derived from previously established confirmative PCR techniques. This similarity in results highlights the method’s accuracy and reliability.

Application and Benefits

  • The simplicity and efficiency of the single-round and multiplex PCR method make it a useful tool for differentiating between B. caballi and B. equi infections. This method can be used in routine diagnostic laboratory settings as well as in wider epidemiological studies, offering a streamlined and cost-effective approach to detecting these equine diseases.

The research paper, thus, provides a comprehensive layout of a simpler and more efficient method for detecting equine blood parasites, notably, Babesia caballi and Babesia equi. This will serve to improve diagnostic procedures in veterinary medicine, especially in areas where these parasites are common.

Cite This Article

APA
Alhassan A, Pumidonming W, Okamura M, Hirata H, Battsetseg B, Fujisaki K, Yokoyama N, 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(1-2), 43-49. https://doi.org/10.1016/j.vetpar.2004.12.018

Publication

Veterinary parasitology
ISSN: 0304-4017
NlmUniqueID: 7602745
Country: Netherlands
Language: English
Volume: 129
Issue: 1-2
Pages: 43-49

Researcher Affiliations

Alhassan, Andy
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Molecular Center for ProtoZoan, Obihiro, Hokkaido 080-8555, Japan.
Pumidonming, Wilawan
    Okamura, Masashi
      Hirata, Haruyuki
        Battsetseg, Badgar
          Fujisaki, Kozo
            Yokoyama, Naoaki
              Igarashi, Ikuo

                MeSH Terms

                • Animals
                • Antibodies, Protozoan / blood
                • Babesia / genetics
                • Babesia / isolation & purification
                • Babesiosis / blood
                • Babesiosis / diagnosis
                • Babesiosis / veterinary
                • DNA, Protozoan / blood
                • Horse Diseases / blood
                • Horse Diseases / diagnosis
                • Horses
                • In Vitro Techniques
                • Molecular Weight
                • Polymerase Chain Reaction / methods
                • Polymerase Chain Reaction / veterinary
                • RNA, Ribosomal, 18S / genetics
                • Sensitivity and Specificity
                • Sequence Alignment / veterinary
                • Species Specificity

                Citations

                This article has been cited 49 times.
                1. Mahdy OA, Nassar AM, Elsawy BSM, Alzan HF, Kandil OM, Mahmoud MS, Suarez CE. Cross-sectional analysis of Piroplasma species-infecting camel (Camelus dromedaries) in Egypt using a multipronged molecular diagnostic approach. Front Vet Sci 2023;10:1178511.
                  doi: 10.3389/fvets.2023.1178511pubmed: 37187929google scholar: lookup
                2. Kumar B, Maharana BR, Thakre B, Brahmbhatt NN, Joseph JP. 18S rRNA Gene-Based Piroplasmid PCR: An Assay for Rapid and Precise Molecular Screening of Theileria and Babesia Species in Animals. Acta Parasitol 2022 Dec;67(4):1697-1707.
                  doi: 10.1007/s11686-022-00625-2pubmed: 36178614google scholar: lookup
                3. Lv K, Zhang Y, Yang Y, Liu Z, Deng L. Development of Nested PCR and Duplex Real-Time Fluorescence Quantitative PCR Assay for the Simultaneous Detection of Theileria equi and Babesia caballi. Front Vet Sci 2022;9:873190.
                  doi: 10.3389/fvets.2022.873190pubmed: 35664851google scholar: lookup
                4. Salinas-Estrella E, Ueti MW, Lobanov VA, Castillo-Payró E, Lizcano-Mata A, Badilla C, Martínez-Ibáñez F, Mosqueda J. Serological and molecular detection of Babesia caballi and Theileria equi in Mexico: A prospective study. PLoS One 2022;17(3):e0264998.
                  doi: 10.1371/journal.pone.0264998pubmed: 35259206google scholar: lookup
                5. Onyiche TE, Igwenagu E, Malgwi SA, Omeh IJ, Biu AA, Thekisoe O. Hematology and biochemical values in equines naturally infected with Theileria equi in Nigeria. Trop Anim Health Prod 2022 Feb 14;54(2):103.
                  doi: 10.1007/s11250-022-03095-wpubmed: 35157179google scholar: lookup
                6. Elsawy BSM, Nassar AM, Alzan HF, Bhoora RV, Ozubek S, Mahmoud MS, Kandil OM, Mahdy OA. Rapid Detection of Equine Piroplasms Using Multiplex PCR and First Genetic Characterization of Theileria haneyi in Egypt. Pathogens 2021 Oct 31;10(11).
                  doi: 10.3390/pathogens10111414pubmed: 34832570google scholar: lookup
                7. Galon EM, Macalanda AM, Garcia MM, Ibasco CJ, Garvida A, Ji S, Zafar I, Hasegawa Y, Liu M, Ybañez RH, Umemiya-Shirafuji R, Ybañez A, Claveria F, Xuan X. Molecular Identification of Selected Tick-Borne Protozoan and Bacterial Pathogens in Thoroughbred Racehorses in Cavite, Philippines. Pathogens 2021 Oct 13;10(10).
                  doi: 10.3390/pathogens10101318pubmed: 34684266google scholar: lookup
                8. Seo HJ, Truong AT, Kim KH, Lim JY, Min S, Kim HC, Yoo MS, Yoon SS, Klein TA, Cho YS. Molecular Detection and Phylogenetic Analysis of Tick-Borne Pathogens in Ticks Collected from Horses in the Republic of Korea. Pathogens 2021 Aug 24;10(9).
                  doi: 10.3390/pathogens10091069pubmed: 34578102google scholar: lookup
                9. Sharma B, R Ganta R, Stone D, Alhassan A, Lanza-Perea M, Matthew Belmar V, Karasek I, Cooksey E, M Butler C, Gibson K, J Wilkerson M. Development of a Multiplex PCR and Magnetic DNA Capture Assay for Detecting Six Species Pathogens of the Genera Anaplasma and Ehrlichia in Canine, Bovine, Caprine and Ovine Blood Samples from Grenada, West Indies. Pathogens 2021 Feb 10;10(2).
                  doi: 10.3390/pathogens10020192pubmed: 33578784google scholar: lookup
                10. Tirosh-Levy S, Gottlieb Y, Fry LM, Knowles DP, Steinman A. Twenty Years of Equine Piroplasmosis Research: Global Distribution, Molecular Diagnosis, and Phylogeny. Pathogens 2020 Nov 8;9(11).
                  doi: 10.3390/pathogens9110926pubmed: 33171698google scholar: lookup
                11. Zhao S, Wang H, Zhang S, Xie S, Li H, Zhang X, Jia L. First report of genetic diversity and risk factor analysis of equine piroplasm infection in equids in Jilin, China. Parasit Vectors 2020 Sep 9;13(1):459.
                  doi: 10.1186/s13071-020-04338-1pubmed: 32907616google scholar: lookup
                12. Onyiche TE, Taioe MO, Ogo NI, Sivakumar T, Biu AA, Mbaya AW, Xuan X, Yokoyama N, Thekisoe O. Molecular evidence of Babesia caballi and Theileria equi in equines and ticks in Nigeria: prevalence and risk factors analysis. Parasitology 2020 Sep;147(11):1238-1248.
                  doi: 10.1017/S0031182020000992pubmed: 32605687google scholar: lookup
                13. Tirosh-Levy S, Steinman A, Levy H, Katz Y, Shtilman M, Gottlieb Y. Parasite load and genotype are associated with clinical outcome of piroplasm-infected equines in Israel. Parasit Vectors 2020 May 20;13(1):267.
                  doi: 10.1186/s13071-020-04133-ypubmed: 32434550google scholar: lookup
                14. Lei R, Wang X, Zhang D, Liu Y, Chen Q, Jiang N. Rapid isothermal duplex real-time recombinase polymerase amplification (RPA) assay for the diagnosis of equine piroplasmosis. Sci Rep 2020 Mar 5;10(1):4096.
                  doi: 10.1038/s41598-020-60997-1pubmed: 32139744google scholar: lookup
                15. Tirosh-Levy S, Gottlieb Y, Mimoun L, Mazuz ML, Steinman A. Transplacental Transmission of Theileria equi Is Not a Common Cause of Abortions and Infection of Foals in Israel. Animals (Basel) 2020 Feb 21;10(2).
                  doi: 10.3390/ani10020341pubmed: 32098113google scholar: lookup
                16. Sunday Idoko I, Tirosh-Levy S, Leszkowicz Mazuz M, Mohammed Adam B, Sikiti Garba B, Wesley Nafarnda D, Steinman A. Genetic Characterization of Piroplasms in Donkeys and Horses from Nigeria. Animals (Basel) 2020 Feb 18;10(2).
                  doi: 10.3390/ani10020324pubmed: 32085574google scholar: lookup
                17. Elata A, Mossaad E, Satti R, Matar N, Ohari Y, Xuan X, Inoue N, Suganuma K. Serological and molecular detection of selected hemoprotozoan parasites in donkeys in West Omdurman, Khartoum State, Sudan. J Vet Med Sci 2020 Mar 5;82(3):286-293.
                  doi: 10.1292/jvms.19-0534pubmed: 31969541google scholar: lookup
                18. Aziz KJ, Al-Barwary LTO. Epidemiological Study of Equine Piroplasmosis (Theileria equi and Babesia caballi) by Microscopic Examination and Competitive-ELISA in Erbil Province North-Iraq. Iran J Parasitol 2019 Jul-Sep;14(3):404-412.
                  pubmed: 31673258
                19. Sant C, Allicock OM, d'Abadie R, Charles RA, Georges K. Phylogenetic analysis of Theileria equi and Babesia caballi sequences from thoroughbred mares and foals in Trinidad. Parasitol Res 2019 Apr;118(4):1171-1177.
                  doi: 10.1007/s00436-019-06240-xpubmed: 30761425google scholar: lookup
                20. Díaz-Sánchez AA, Pires MS, Estrada CY, Cañizares EV, Del Castillo Domínguez SL, Cabezas-Cruz A, Rivero EL, da Fonseca AH, Massard CL, Corona-González B. First molecular evidence of Babesia caballi and Theileria equi infections in horses in Cuba. Parasitol Res 2018 Oct;117(10):3109-3118.
                  doi: 10.1007/s00436-018-6005-5pubmed: 30033488google scholar: lookup
                21. Ionita M, Nicorescu IM, Pfister K, Mitrea IL. Parasitological and molecular diagnostic of a clinical Babesia caballi outbreak in Southern Romania. Parasitol Res 2018 Jul;117(7):2333-2339.
                  doi: 10.1007/s00436-018-5899-2pubmed: 29766270google scholar: lookup
                22. Lobanov VA, Peckle M, Massard CL, Brad Scandrett W, Gajadhar AA. Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis. Parasit Vectors 2018 Mar 2;11(1):125.
                  doi: 10.1186/s13071-018-2751-6pubmed: 29499748google scholar: lookup
                23. Montes-Cortés MG, Fernández-García JL, Martínez-Estéllez MÁH. Genetic Variation of the β-tubulin Gene of Babesia caballi Strains. J Arthropod Borne Dis 2017 Sep;11(3):344-353.
                  pubmed: 29322051
                24. Montes Cortés MG, Fernández-García JL, Habela Martínez-Estéllez MÁ. Seroprevalence of Theileria equi and Babesia caballi in horses in Spain. Parasite 2017;24:14.
                  doi: 10.1051/parasite/2017015pubmed: 28497743google scholar: lookup
                25. Abedi V, Razmi G, Seifi H, Naghibi A. Molecular detection of equine piroplasms in donkeys (Equus asinus) in North Khorasan province, Iran. Iran J Vet Res 2015 Spring;16(2):202-4.
                  pubmed: 27175176
                26. Sumbria D, Das Singla L, Sharma A. Theileria equi and Babesia caballi infection of equids in Punjab, India: a serological and molecular survey. Trop Anim Health Prod 2016 Jan;48(1):45-52.
                  doi: 10.1007/s11250-015-0917-1pubmed: 26387094google scholar: lookup
                27. Nourollahi-Fard SR, Khalili M, Ghalekhani N. Detection of Theileria annulata in blood samples of native cattle by PCR and smear method in Southeast of Iran. J Parasit Dis 2015 Jun;39(2):249-52.
                  doi: 10.1007/s12639-013-0333-2pubmed: 26064010google scholar: lookup
                28. Junlong L, Li Y, Liu A, Guan G, Xie J, Yin H, Luo J. Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle. Parasitol Res 2015 Jul;114(7):2715-21.
                  doi: 10.1007/s00436-015-4478-zpubmed: 25895064google scholar: lookup
                29. Mans BJ, Pienaar R, Latif AA. A review of Theileria diagnostics and epidemiology. Int J Parasitol Parasites Wildl 2015 Apr;4(1):104-18.
                  doi: 10.1016/j.ijppaw.2014.12.006pubmed: 25830110google scholar: lookup
                30. Malekifard F, Tavassoli M, Yakhchali M, Darvishzadeh R. Detection of Theileria equi and Babesia caballi using microscopic and molecular methods in horses in suburb of Urmia, Iran. Vet Res Forum 2014 Spring;5(2):129-33.
                  pubmed: 25568706
                31. Mosqueda J, Olvera-Ramirez A, Aguilar-Tipacamu G, Canto GJ. Current advances in detection and treatment of babesiosis. Curr Med Chem 2012;19(10):1504-18.
                  doi: 10.2174/092986712799828355pubmed: 22360483google scholar: lookup
                32. Niu Q, Luo J, Guan G, Ma M, Liu Z, Liu A, Dang Z, Gao J, Ren Q, Li Y, Liu J, Yin H. Detection and differentiation of ovine Theileria and Babesia by reverse line blotting in China. Parasitol Res 2009 Jun;104(6):1417-23.
                  doi: 10.1007/s00436-009-1344-xpubmed: 19205742google scholar: lookup
                33. Salim BO, Hassan SM, Bakheit MA, Alhassan A, Igarashi I, Karanis P, Abdelrahman MB. Diagnosis of Babesia caballi and Theileria equi infections in horses in Sudan using ELISA and PCR. Parasitol Res 2008 Oct;103(5):1145-50.
                  doi: 10.1007/s00436-008-1108-zpubmed: 18618143google scholar: lookup
                34. Alhassan A, Iseki H, Kim C, Yokoyama N, Igarashi I. Comparison of polymerase chain reaction methods for the detection of Theileria equi infection using whole blood compared with pre-extracted DNA samples as PCR templates. Trop Anim Health Prod 2007 Jun;39(5):369-74.
                  doi: 10.1007/s11250-007-9025-1pubmed: 17944307google scholar: lookup
                35. Heim A, Passos LM, Ribeiro MF, Costa-Júnior LM, Bastos CV, Cabral DD, Hirzmann J, Pfister K. Detection and molecular characterization of Babesia caballi and Theileria equi isolates from endemic areas of Brazil. Parasitol Res 2007 Dec;102(1):63-8.
                  doi: 10.1007/s00436-007-0726-1pubmed: 17828553google scholar: lookup
                36. Alhassan A, Govind Y, Tam NT, Thekisoe OM, Yokoyama N, Inoue N, Igarashi I. Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis. Parasitol Res 2007 Apr;100(5):1165-8.
                  doi: 10.1007/s00436-006-0430-6pubmed: 17216488google scholar: lookup
                37. Qin S, Kulabieke T, Mizhamuhan D, Zhang M, Jin M, Abula G, Pi M, Wang H, Zhang Y, Guo Q. Molecular Prevalence and Genotypic Diversity of Theileria equi in Xinjiang, China, Based on Three Genes. Vet Sci 2025 Dec 25;13(1).
                  doi: 10.3390/vetsci13010027pubmed: 41600683google scholar: lookup
                38. Soliman AM, Elhawary NM, Helmy NM, El-Seify MA, Amer MM, Mohamed S, Memon FU, Rashid MHO, Gadelhaq SM. 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. Iran J Vet Res 2025;26(2):145-151.
                  doi: 10.22099/ijvr.2025.51028.7553pubmed: 41170307google scholar: lookup
                39. Hacilarlioglu S, Bilgic HB, Karagenc T, Aydin HB, Toker H, Kanlioglu H, Pekagirbas M, Bakirci S. Molecular Detection and Prevalence of Equine Piroplasmosis and Other Blood Parasites in Equids of Western Aegean Türkiye. Vet Sci 2025 Aug 27;12(9).
                  doi: 10.3390/vetsci12090826pubmed: 41012752google scholar: lookup
                40. Sadeddine R, Righi S, Saidani K, Benakhla A. First Molecular Characterization of Theileria equi from Northeastern Algeria. Acta Parasitol 2025 Mar 6;70(2):66.
                  doi: 10.1007/s11686-025-01006-1pubmed: 40050535google scholar: lookup
                41. Facile V, Magliocca M, Dini FM, Imposimato I, Mariella J, Freccero F, Urbani L, Rinnovati R, Sel E, Gallina L, Castagnetti C, Galuppi R, Battilani M, Balboni A. Molecular Diagnosis and Identification of Equine Piroplasms: Challenges and Insights from a Study in Northern Italy. Animals (Basel) 2025 Feb 5;15(3).
                  doi: 10.3390/ani15030437pubmed: 39943207google scholar: lookup
                42. Aziz KJ, Barwary LTOA, Issa NA, Abdulwahid MJ. Prevalence of Theileria Equi and Babesia Caballi and Associated Hemato-Biochemical Changes in Racing Horses in Erbil Province, Iraq. Acta Parasitol 2025 Jan 24;70(1):38.
                  doi: 10.1007/s11686-024-00987-9pubmed: 39853481google scholar: lookup
                43. Ulucesme MC, Ozubek S, Aktas M. Development and Evaluation of a Semi-Nested PCR Method Based on the 18S ribosomal RNA Gene for the Detection of Babesia aktasi Infections in Goats. Vet Sci 2024 Oct 1;11(10).
                  doi: 10.3390/vetsci11100466pubmed: 39453058google scholar: lookup
                44. Wu X, Xu J, Su L, Li E, Wang S, Hornok S, Liu G, Wang Y. First Molecular Evidence of Babesia caballi and Theileria equi in Imported Donkeys from Kyrgyzstan. Pathogens 2024 Aug 23;13(9).
                  doi: 10.3390/pathogens13090713pubmed: 39338905google scholar: lookup
                45. Ochi A, Toya Y, Sengoku M, Tsuchiya S, Kishi D, Ueno T. In vitro evaluation of the automated hematology analyzer XN-31 for rapid diagnosis of equine piroplasmosis. Microbiol Spectr 2024 Oct 3;12(10):e0058224.
                  doi: 10.1128/spectrum.00582-24pubmed: 39269182google scholar: lookup
                46. Mohammad-Naseri A, Shokrani H, Rahmani-Shahraki A. Equine Piroplasmosis in Asymptomatic Horses of Western Iran: Comparison of Microscopic Examination and Multiplex PCR. Acta Parasitol 2024 Mar;69(1):813-818.
                  doi: 10.1007/s11686-024-00804-3pubmed: 38424400google scholar: lookup
                47. Ma Y, Jian Y, Wang G, Li X, Wang G, Hu Y, Yokoyama N, Ma L, Xuan X. Molecular Identification of Babesia and Theileria Infections in Livestock in the Qinghai-Tibetan Plateau Area, China. Animals (Basel) 2024 Feb 1;14(3).
                  doi: 10.3390/ani14030476pubmed: 38338119google scholar: lookup
                48. Ahedor B, Otgonsuren D, Zhyldyz A, Guswanto A, Ngigi NMM, Valinotti MFR, Kothalawala H, Kalaichelvan N, Silva SSP, Kothalawala H, Acosta TJ, Sivakumar T, Yokoyama N. Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes. Parasit Vectors 2023 Nov 25;16(1):435.
                  doi: 10.1186/s13071-023-06045-zpubmed: 38007442google scholar: lookup
                49. Malgwi SA, Ogunsakin RE, Oladepo AD, Adeleke MA, Okpeku M. A Forty-Year Analysis of the Literature on Babesia Infection (1982-2022): A Systematic Bibliometric Approach. Int J Environ Res Public Health 2023 Jun 16;20(12).
                  doi: 10.3390/ijerph20126156pubmed: 37372744google scholar: lookup
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