FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Frontiers in veterinary science2022; 9; 873190; doi: 10.3389/fvets.2022.873190

Development of Nested PCR and Duplex Real-Time Fluorescence Quantitative PCR Assay for the Simultaneous Detection of Theileria equi and Babesia caballi.

Abstract: Equine piroplasmosis (EP) is a type of blood protozoan disease caused by tick-borne parasites, (), () and . While many studies have been conducted on EP diagnosis, diagnostic methods exhibiting high sensitivity and specificity remain lacking. Therefore, nested PCR (nPCR) and duplex real-time fluorescence quantitative PCR (qPCR) that can simultaneously detect both and causing agents were established and compared. The two techniques were used to analyze 36 horse blood samples for EP. This set of samples was also detected by a multinested PCR (mnPCR) targeting the gene of and the gene of . By nPCR, duplex real-time fluorescence qPCR and mnPCR, infections with were detected in 16.67% (6/36), 2.78% (1/36), 19.44% (7/36) of the horses, respectively. The prevalence was 58.33% (21/36) by the nPCR, 33.33% (12/36) by the duplex real-time fluorescence qPCR and 2.78% (1/36) by the mnPCR. The overall prevalence of infection with mixed parasites by nPCR was 5.56% (2/36), by duplex real-time fluorescence qPCR was 2.78% (1/36) and by mnPCR 0% (0/36). Results suggest that nPCR can detect and positive samples with good specificity and sensitivity, although distinguishing between the two parasites requires an electrophoresis with 4% agarose gels. The duplex real-time fluorescence qPCR can readily distinguish between and infection, but with low sensitivity.
Copyright © 2022 Lv, Zhang, Yang, Liu and Deng.
Get Full Text
Publication Date: 2022-05-18 PubMed ID: 35664851PubMed Central: PMC9158504DOI: 10.3389/fvets.2022.873190Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

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 article focuses on the development of nested Polymerase Chain Reactions (nPCR) and duplex real time fluorescence quantitative PCR (qPCR) for the simultaneous detection of Theileria equi and Babesia caballi, which cause Equine piroplasmosis, a blood protozoan disease. The study found that nPCR has good sensitivity and specificity, while duplex real-time fluorescence qPCR can distinguish between the two parasites.

Objective of the Research

  • The main purpose of this research is to develop nPCR and duplex real-time fluorescence qPCR that can simultaneously detect both Theileria equi and Babesia caballi.
  • The comparative effectiveness and efficiency of these tests were assessed. This was done by detecting the level of sensitivity and specificity of both on a set of horse blood samples.
  • The objective was also to overcome the gap left by lack of diagnostic methods exhibiting high sensitivity and specificity for Equine piroplasmosis (EP).

Methodology

  • The researchers used nPCR and duplex real-time fluorescence qPCR techniques to analyze 36 horse blood samples for EP.
  • These samples were also tested by a multinested PCR (mnPCR) targeting specific genes of Theileria equi and Babesia caballi.
  • The detection rates were compared across all three methods.

Results

  • nPCR detected infections with Theileria equi in 16.67% (6/36) of the horses, while duplex real-time fluorescence qPCR detected infections in 2.78% (1/36), and mnPCR detected infections in 19.44% (7/36).
  • Babesia caballi prevalence was 58.33% (21/36) by the nPCR, 33.33% (12/36) by the duplex real-time fluorescence qPCR and 2.78% (1/36) by the mnPCR.
  • The overall prevalence of infection with mixed parasites by nPCR was 5.56% (2/36), by duplex real-time fluorescence qPCR was 2.78% (1/36) and by mnPCR 0% (0/36).
  • nPCR demonstrated good specificity and sensitivity, although distinguishing between the two parasites required electrophoresis with 4% agarose gels.
  • The duplex real-time fluorescence qPCR method was able to distinguish between Theileria equi and Babesia caballi infection. However, it demonstrated a low level of sensitivity.

Conclusion

  • The research demonstrates that nPCR can accurately and specifically detect Theileria equi and Babesia caballi in horse blood samples although an additional process (electrophoresis with 4% agarose gels) is required to differentiate between the two.
  • The duplex real-time fluorescence qPCR, despite its lower sensitivity, could readily distinguish between these two infections, indicating the method has potential for further development and refinement.

Cite This Article

APA
Lv K, Zhang Y, Yang Y, Liu Z, Deng L. (2022). 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, 9, 873190. https://doi.org/10.3389/fvets.2022.873190

Publication

Frontiers in veterinary science
ISSN: 2297-1769
NlmUniqueID: 101666658
Country: Switzerland
Language: English
Volume: 9
Pages: 873190
PII: 873190

Researcher Affiliations

Lv, Kunying
  • Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.
Zhang, Yiwei
  • Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, Key Laboratory of Zoonosis, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.
Yang, Yixin
  • Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.
Liu, Zheng
  • Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, Key Laboratory of Zoonosis, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.
Deng, Liang
  • Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

This article includes 31 references
  1. Guidi E, Pradier S, Lebert I, Leblond A. Piroplasmosis in an endemic area: analysis of the risk factors and their implications in the control of Theileriosis and Babesiosis in horses.. Parasitol Res 2015 Jan;114(1):71-83.
    doi: 10.1007/s00436-014-4161-9pubmed: 25280516google scholar: lookup
  2. Knowles DP, Kappmeyer LS, Haney D, Herndon DR, Fry LM, Munro JB, Sears K, Ueti MW, Wise LN, Silva M, Schneider DA, Grause J, White SN, Tretina K, Bishop RP, Odongo DO, Pelzel-McCluskey AM, Scoles GA, Mealey RH, Silva JC. 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 2018 Aug;48(9-10):679-690.
    doi: 10.1016/j.ijpara.2018.03.010pubmed: 29885436google scholar: lookup
  3. Elelu N. Tick-borne relapsing fever as a potential veterinary medical problem.. Vet Med Sci 2018 Nov;4(4):271-279.
    doi: 10.1002/vms3.108pmc: PMC6236141pubmed: 29943903google scholar: lookup
  4. Short MA, Clark CK, Harvey JW, Wenzlow N, Hawkins IK, Allred DR, Knowles DP, Corn JL, Grause JF, Hennager SG, Kitchen DL, Traub-Dargatz JL. Outbreak of equine piroplasmosis in Florida.. J Am Vet Med Assoc 2012 Mar 1;240(5):588-95.
    doi: 10.2460/javma.240.5.588pubmed: 22332629google scholar: lookup
  5. 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/pathogens9110926pmc: PMC7695325pubmed: 33171698google scholar: lookup
  6. Kerber CE, Ferreira F, Pereira MC. Control of equine piroplasmosis in Brazil.. Onderstepoort J Vet Res 1999 Jun;66(2):123-7.
    pubmed: 10486829
  7. Lempereur L, Beck R, Fonseca I, Marques C, Duarte A, Santos M, Zúquete S, Gomes J, Walder G, Domingos A, Antunes S, Baneth G, Silaghi C, Holman P, Zintl A. Guidelines for the Detection of Babesia and Theileria Parasites.. Vector Borne Zoonotic Dis 2017 Jan;17(1):51-65.
    doi: 10.1089/vbz.2016.1955pubmed: 28055573google scholar: lookup
  8. Bashiruddin JB, Cammà C, Rebêlo E. Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16S rRNA gene.. Vet Parasitol 1999 Jul;84(1-2):75-83.
    doi: 10.1016/S0304-4017(99)00049-7pubmed: 10435792google scholar: lookup
  9. Nicolaiewsky TB, Richter MF, Lunge VR, Cunha CW, Delagostin O, Ikuta N, Fonseca AS, da Silva SS, Ozaki LS. Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction.. Vet Parasitol 2001 Oct 31;101(1):9-21.
    doi: 10.1016/S0304-4017(01)00471-Xpubmed: 11587829google scholar: lookup
  10. Xuan X, Larsen A, Ikadai H, Tanaka T, Igarashi I, Nagasawa H, Fujisaki K, Toyoda Y, Suzuki N, Mikami T. Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.. J Clin Microbiol 2001 Feb;39(2):705-9.
    doi: 10.1128/JCM.39.2.705-709.2001pmc: PMC87800pubmed: 11158131google scholar: lookup
  11. Ueti MW, Palmer GH, Kappmeyer LS, Scoles GA, Knowles DP. Expression of equi merozoite antigen 2 during development of Babesia equi in the midgut and salivary gland of the vector tick Boophilus microplus.. J Clin Microbiol 2003 Dec;41(12):5803-9.
    doi: 10.1128/JCM.41.12.5803-5809.2003pmc: PMC308990pubmed: 14662988google scholar: lookup
  12. Bhoora R, Quan M, Zweygarth E, Guthrie AJ, Prinsloo SA, Collins NE. Sequence heterogeneity in the gene encoding the rhoptry-associated protein-1 (RAP-1) of Babesia caballi isolates from South Africa.. Vet Parasitol 2010 May 11;169(3-4):279-88.
    doi: 10.1016/j.vetpar.2010.01.009pubmed: 20138703google scholar: lookup
  13. Ikadai H, Xuan X, Igarashi I, Tanaka S, Kanemaru T, Nagasawa H, Fujisaki K, Suzuki N, Mikami T. Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay.. J Clin Microbiol 1999 Nov;37(11):3475-80.
    doi: 10.1128/JCM.37.11.3475-3480.1999pmc: PMC85671pubmed: 10523537google scholar: lookup
  14. Brüning A, Phipps P, Posnett E, Canning EU. Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis.. Vet Parasitol 1997 Jan;68(1-2):11-26.
    doi: 10.1016/S0304-4017(96)01074-6pubmed: 9066047google scholar: lookup
  15. Montes Cortés MG, Fernández-García JL, Habela Martínez-Estéllez MÁ. A multinested PCR for detection of the equine piroplasmids Babesia caballi and Theileria equi.. Ticks Tick Borne Dis 2019 Feb;10(2):305-313.
    doi: 10.1016/j.ttbdis.2018.11.008pubmed: 30472099google scholar: lookup
  16. Alhassan A, Pumidonming W, Okamura M, Hirata H, Battsetseg B, Fujisaki K, Yokoyama N, Igarashi I. Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood.. Vet Parasitol 2005 Apr 20;129(1-2):43-9.
    doi: 10.1016/j.vetpar.2004.12.018pubmed: 15817201google scholar: lookup
  17. Mahmoud MS, El-Ezz NT, Abdel-Shafy S, Nassar SA, El Namaky AH, Khalil WK, Knowles D, Kappmeyer L, Silva MG, Suarez CE. Assessment of Theileria equi and Babesia caballi infections in equine populations in Egypt by molecular, serological and hematological approaches.. Parasit Vectors 2016 May 4;9:260.
    doi: 10.1186/s13071-016-1539-9pmc: PMC4857240pubmed: 27146413google scholar: lookup
  18. 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.
    pmc: PMC4279624pubmed: 25568706
  19. Rampersad J, Cesar E, Campbell MD, Samlal M, Ammons D. A field evaluation of PCR for the routine detection of Babesia equi in horses.. Vet Parasitol 2003 May 30;114(2):81-7.
    doi: 10.1016/S0304-4017(03)00129-8pubmed: 12781470google scholar: lookup
  20. Nardini R, Bartolomé Del Pino LE, Cersini A, Manna G, Viola MR, Antognetti V, Autorino GL, Scicluna MT. Comparison of PCR-based methods for the detection of Babesia caballi and Theileria equi in field samples collected in Central Italy.. Parasitol Res 2021 Jun;120(6):2157-2164.
    doi: 10.1007/s00436-021-07153-4pubmed: 33855619google scholar: lookup
  21. Bhoora R, Quan M, Matjila PT, Zweygarth E, Guthrie AJ, Collins NE. Sequence heterogeneity in the equi merozoite antigen gene (ema-1) of Theileria equi and development of an ema-1-specific TaqMan MGB assay for the detection of T. equi.. Vet Parasitol 2010 Aug 27;172(1-2):33-45.
    doi: 10.1016/j.vetpar.2010.04.025pubmed: 20493635google 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/s13071-018-2751-6pmc: PMC5834856pubmed: 29499748google scholar: lookup
  23. 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
  24. Kim CM, Blanco LB, Alhassan A, Iseki H, Yokoyama N, Xuan X, Igarashi I. Diagnostic real-time PCR assay for the quantitative detection of Theileria equi from equine blood samples.. Vet Parasitol 2008 Feb 14;151(2-4):158-63.
    doi: 10.1016/j.vetpar.2007.10.023pubmed: 18077095google scholar: lookup
  25. Bhoora RV, Pienaar R, Cornelius F, Josemans A, Matthee O, Marumo R, Troskie C, Mans BJ. Multiplex hydrolysis-probe assay for the simultaneous detection of Theileria equi and Babesia caballi infections in equids.. Vet Parasitol 2018 May 15;255:61-68.
    doi: 10.1016/j.vetpar.2018.03.022pubmed: 29773138google scholar: lookup
  26. Bhoora R, Quan M, Franssen L, Butler CM, van der Kolk JH, Guthrie AJ, Zweygarth E, Jongejan F, Collins NE. 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 2010 Mar 25;168(3-4):201-11.
    doi: 10.1016/j.vetpar.2009.11.011pubmed: 20031328google scholar: lookup
  27. Henneke DR, Potter GD, Kreider JL, Yeates BF. Relationship between condition score, physical measurements and body fat percentage in mares.. Equine Vet J 1983 Oct;15(4):371-2.
    doi: 10.1111/j.2042-3306.1983.tb01826.xpubmed: 6641685google scholar: lookup
  28. 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/s41598-020-60997-1pmc: PMC7058082pubmed: 32139744google scholar: lookup
  29. Hirata H, Xuan X, Yokoyama N, Nishikawa Y, Fujisaki K, Suzuki N, Igarashi I. Identification of a specific antigenic region of the P82 protein of Babesia equi and its potential use in serodiagnosis.. J Clin Microbiol 2003 Feb;41(2):547-51.
    doi: 10.1128/JCM.41.2.547-551.2003pmc: PMC149686pubmed: 12574244google scholar: lookup
  30. Böse R, Jorgensen WK, Dalgliesh RJ, Friedhoff KT, de Vos AJ. Current state and future trends in the diagnosis of babesiosis.. Vet Parasitol 1995 Mar;57(1-3):61-74.
    doi: 10.1016/0304-4017(94)03111-9pubmed: 7597794google scholar: lookup
  31. 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/pathogens10111414pmc: PMC8620363pubmed: 34832570google scholar: lookup

Citations

This article has been cited 2 times.
  1. Pan Y, Yu Q, Wang Q, Li Q, Tian W, Xin L, Hu X, Xiao H, Liu Y, Zhu LRD, Lan L, Zhu L, Wu J. Establishment and epidemiological investigation of a dual fluorescent qPCR assay for Pasteurella multocida and Salmonella in yaks in the Tibetan Autonomous Prefecture of Garzê, China. Front Cell Infect Microbiol 2025;15:1599817.
    doi: 10.3389/fcimb.2025.1599817pubmed: 40535542google scholar: lookup
  2. 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
Subscribe to our newsletter
Join 100,127 horse owners like you who receive our email updates on horse health and nutrition.