A pilot study to evaluate the consistency of different laboratory diagnostic methods for detecting Babesia spp. in horses

Equine piroplasmosis remains relevant in veterinary practice owing to the possibility of acute disease and latent carrier status, in which laboratory detection of the pathogen is difficult. The aim of the study was a pilot assessment of agreement between blood smear microscopy, local screening PCR and laboratory real-time PCR for the detection of Babesia spp. in horses of different clinical status. Fourteen animals were examined. All underwent clinical examination, microscopy of thin peripheral blood smears, screening PCR using a commercial kit for the detection of Babesia spp. DNA, and testing in an accredited veterinary laboratory, the results of which were used as the comparator method. The full protocol of the laboratory method was not disclosed, therefore it was used as a comparator rather than an absolute reference method. Clinical signs compatible with acute infestation were recorded in 3 of 14 animals, in which the pathogen was detected by microscopy and screening PCR. Laboratory PCR was positive in 10 of 14 horses, including clinically healthy animals negative by the routine methods. Positive percent agreement of microscopy and local screening PCR with the comparator was 30,0 % each, negative percent agreement was 100,0 and 75,0 %, respectively. Cohen kappa was 0,196 and 0,034, indicating slight agreement. Values are reported with 95 % confidence intervals calculated by the Wilson method. A negative result of the routine methods does not exclude Babesia spp. infestation in clinically healthy horses. The findings are preliminary and require verification in an expanded sample using a validated reference method.

1. Marchenko, V. A. Ixodid infestations of horses, seasonal dynamics and piroplasmida infection of pasture ticks in the Gorny Altai/ V. A. Marchenko, V. A. Rar, I. V. Biryukov // Russian Journal of Parasitology, Russian Journal of Parasitology. 2023;17(3):352—364. DOI 10.31016/1998-8435-2023-17-3-352-364 (In Russ.).

2. Tirosh-Levy S., Gottlieb Y., Fry L.M., Knowles D.P., Steinman A. Twenty Years of Equine Piroplasmosis Research: Global Distribution, Molecular Diagnosis, and Phylogeny. Pathogens. 2020;9(11):926. DOI: 10.3390/pathogens9110926.

3. Onyiche T. E., Taioe M. O., Molefe N. I., Biu A. A., Luka J., Omeh I. J., Yokoyama N., Thekisoe O. M. M. Equine piroplasmosis: an insight into global exposure of equids from 1990 to 2019 by systematic review and meta—analysis. Parasitology. 2020;147(13):1411—1424. DOI: 10.1017/s0031182020001407

4. Rar V., Marchenko V., Suntsova O., Epikhina T., Tikunov A., Meltsov I., Fedorets V., Igolkina Y., Kozlova I., Tikunova N. The first study of the prevalence and genetic diversity of Theileria equi and Babesia caballi in horses in Russia. Parasitology Research. 2024;123:279. DOI: 10.1007/s00436-024-08300-3.

5. Onyiche T. E., Suganuma K., Igarashi I., Yokoyama N., Xuan X., Thekisoe O. M. M. A Review on Equine Piroplasmosis: Epidemiology, Vector Ecology, Risk Factors, Host Immunity, Diagnosis and Control. International Journal of Environmental Research and Public Health. 2019;16(10):1736. DOI: 10.3390/ijerph16101736.

6. Onyiche T. E., Taioe M. O., Molefe N. I., Biu A. A., Luka J., Omeh I. J., Yokoyama N., Thekisoe O. New insights in the diagnosis and treatment of equine piroplasmosis: pitfalls, idiosyncrasies, and myths. Frontiers in Veterinary Science. 2024;11:1459989. DOI: 10.3389/fvets.2024.1459989.

7. 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. Frontiers in Veterinary Science. 2022;9:873190. DOI: 10.3389/fvets.2022.873190.

8. Nardini R., Bartolomé Del Pino L.E., Cersini A., Manna G., Viola M.R., Antognetti V., Autorino G.L., Scicluna M.T. Comparison of PCR—based methods for the detection of Babesia caballi and Theileria equi in field samples collected in Central Italy. Parasitology Research. 2021;120:2157—2164. DOI: 10.1007/s00436-021-07153-4.

9. Venter A., Vorster I., Nkosi N.F., Sibeko—Matjila K.P., Bhoora R. Molecular genotyping of Babesia caballi. Veterinary Parasitology. 2024;329:110214. DOI: 10.1016/j.vetpar.2024.110214.

10. Demkina O. V., Helminthological status of sport and breeding horses exported from the Russian Federation / I. A. Solovyeva, G. A. Bondarenko, T. I. Trukhina // International Veterinary Bulletin. 2023;(3):74—78. DOI: 10.52419/issn2072-2419.2023.3.74 (In Russ.).

11. Mège M., Bonsergent C., Viry L., Dhune M., Lecollinet S., Malandrin L. Genetic diversity of equine piroplasmosis agents in Guadeloupe (Caribbeans): first report of Theileria haneyi, evaluation of diagnostic tools and impact of horse movement. Ticks and Tick-borne Diseases. 2025;16(6):102547. DOI: 10.1016/j.ttbdis.2025.102547.

12. Bishop R.P., Kappmeyer L.S., Onzere C., Odongo D., Githaka N., Sears K.P., Knowles D.P., Fry L.M. Equid infective Theileria cluster in distinct 18S rRNA gene clades comprising multiple taxa with unusually broad mammalian host ranges. Parasites & Vectors. 2020;13:261. DOI: 10.1186/s13071-020-04131-0.

13. Giubega S., Dreghiciu C., Ilie M.S., Dărăbuş G. Diagnostic methods used for the detection of Theileria equi: review of the last decade. Cluj Veterinary Journal. 2022;27(2):16—24. DOI: 10.52331/cvj.v27i2.37.

14. Atabek B., Zhyldyz A., Aitakin K., Rysbek N., Jailobek O., Ahedor B., Mumbi N. N. M., Ma Y., Otgonsuren D., Perera W., Guswanto A., Sivakumar T., Yokoyama N. Molecular prevalence and genotypic diversity of Theileria equi and Babesia caballi infecting horses in Kyrgyzstan. Parasitology International. 2024;102:102915. DOI: 10.1016/j.parint.2024.102915.

15. Landis J.R., Koch G.G. The measurement of observer agreement for categorical data. Biometrics. 1977;33(1):159—174. DOI: 10.2307/2529310.

16. Lobanov V. A., Peckle M., Massard C. L., Scandrett W. B., Gajadhar A. A. Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis. Parasites & Vectors. 2018;11(1):125. DOI: 10.1186/s13071-018-2751-6.

17. Dorrego A., Camino E., Gago P., Buendia-Andres A., Acurio K., Gonzalez S., de Juan L., Cruz-Lopez F. Haemato-biochemical characterization of equine piroplasmosis asymptomatic carriers and seropositive, real-time PCR negative horses. Veterinary Parasitology. 2023;323:110046. DOI: 10.1016/j.vetpar.2023.110046.

18. Salinas-Estrella E., Ueti M.W., Lobanov V.A., CastilloPayró 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.0264998.