FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Parasit Vectors / Development and validation of a duplex real-time PCR assay f...
Parasites & vectors2018; 11(1); 125; doi: 10.1186/s13071-018-2751-6

Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis.

Abstract: Equine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay's utility for regulatory testing. We developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay's performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5-90.7%), 90.5% (95% CI: 87.3-92.3%) and 87.4% (95% CI: 84.0-90.2%) of the horses, respectively. The B. caballi prevalence estimates were 9.3% (95% CI: 6.9-12.4%) by the duplex qPCR and 7.9% (95% CI: 5.7-10.9%) by the respective single-target qPCR assay. These values were markedly lower compared to the seroprevalence of 58.6% (95% CI: 53.9-63.2%) obtained by B. caballi-specific cELISA. The relative diagnostic sensitivity of the duplex qPCR for T. equi was 95.5%, as 359 of the 376 horses with exposure to T. equi confirmed by cELISA had parasitemia levels above the detection limit of the molecular assay. In contrast, only 39 (15.5%) of the 252 horses with detectable B. caballi-specific antibodies were positive for this piroplasm species by the duplex qPCR. The duplex qPCR described here performed comparably to the existing single-target qPCR assays for T. equi and B. caballi and will be more cost-effective in terms of results turnaround time and reagent costs when both pathogens are being targeted for disease control and epidemiological investigations. These validation data also support the reliability of the ema-1 gene-specific oligonucleotides developed in this study for confirmatory testing of non-negative serological test results for T. equi by qPCR. However, the B. caballi-specific qPCR cannot be similarly recommended as a confirmatory assay for routine regulatory testing due to the low level of agreement with serological test results demonstrated in this study. Further studies are needed to determine the transmission risk posed by PCR-negative equines with detectable antibodies to B. caballi.
Get Full Text
Publication Date: 2018-03-02 PubMed ID: 29499748PubMed Central: PMC5834856DOI: 10.1186/s13071-018-2751-6Google 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
  • Research Support
  • Non-U.S. Gov't
  • Validation Study

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.

This research paper discusses the development and validation of a new method to diagnose equine piroplasmosis, a significant infection in horses caused by the tick-borne protozoa Theileria equi and Babesia caballi. The researchers developed a rapid, sensitive test, a real-time quantitative PCR (qPCR) assay, which effectively identified both causes of the disease in horse samples and was found to be more cost-effective than existing methods. The test was reliable in confirming results of serological tests for T. equi but not for B. caballi, warranting further research.

Development and Evaluation of The Assay

  • The researchers developed a duplex qPCR assay that targets two specific genes, ema-1 gene of T. equi and the 18S rRNA gene of B. caballi, which are unique to each protozoa.
  • The aim was to simultaneously detect both causative agents of equine piroplasmosis, enhancing testing efficiency and accuracy.
  • After the development, the assay underwent a series of evaluations to determine its sensitivity and reliability.

Results of The Validation Process

  • The researchers ran the duplex qPCR test on samples from 362 horses from Canada and the United States, found negative for equine piroplasmosis using the enzyme-linked immunosorbent assay (cELISA). The duplex qPCR did not yield any false-positive results, indicating its high specificity.
  • Further validation occurred using horse samples from an endemic area in Brazil. The results of the qPCR were compared to two alternative tests; an 18S rRNA qPCR for T. equi developed in Japan, and another 18S rRNA qPCR for B. caballi.
  • The reliability of the cELISA was also assessed by comparing its results to those of the duplex qPCR.
  • Results from the Brazilian horse samples yielded higher infection rates with both protozoa, with all three tests (duplex qPCR, single-target qPCR, and cELISA) producing similar results for T. equi. However, for B. caballi, there was a significant discrepancy between the duplex qPCR/single-target qPCR and the cELISA.

Implications of The Findings

  • The duplex qPCR performed comparably to existing single-target qPCR assays for both T. equi and B. caballi in terms of detection efficiency, but was found to be more cost-effective in terms of response time and reagent costs, making it a good choice for disease control and investigations.
  • Unfortunately, the qPCR test did not correlate well with the cELISA results when testing for B. caballi, suggesting it should not be used as a standalone confirmatory test for this protozoa.
  • Though the new test is highly effective for T. equi detection, the research affirms further investigation is needed to understand the transmission risk of B. caballi in horses that test negative through PCR but possess detectable antibodies.

Cite This Article

APA
Lobanov VA, Peckle M, Massard CL, Brad Scandrett W, Gajadhar AA. (2018). Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis. Parasit Vectors, 11(1), 125. https://doi.org/10.1186/s13071-018-2751-6

Publication

Parasites & vectors
ISSN: 1756-3305
NlmUniqueID: 101462774
Country: England
Language: English
Volume: 11
Issue: 1
Pages: 125
PII: 125

Researcher Affiliations

Lobanov, Vladislav A
  • Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada. Vladislav.Lobanov@inspection.gc.ca.
Peckle, Maristela
  • Department of Animal Parasitology, Veterinary Institute, Federal Rural University of Rio de Janeiro, Seropedica, Rio de Janeiro, Brazil.
Massard, Carlos L
  • Department of Animal Parasitology, Veterinary Institute, Federal Rural University of Rio de Janeiro, Seropedica, Rio de Janeiro, Brazil.
Brad Scandrett, W
  • Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada.
Gajadhar, Alvin A
  • Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada.

MeSH Terms

  • Animals
  • Babesia / genetics
  • Babesia / isolation & purification
  • Babesiosis / diagnosis
  • Babesiosis / epidemiology
  • Babesiosis / parasitology
  • Brazil / epidemiology
  • Canada / epidemiology
  • Enzyme-Linked Immunosorbent Assay / veterinary
  • Horse Diseases / diagnosis
  • Horse Diseases / parasitology
  • Horses
  • Japan / epidemiology
  • Protozoan Infections, Animal / diagnosis
  • Protozoan Infections, Animal / epidemiology
  • RNA, Ribosomal, 18S / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / veterinary
  • Reproducibility of Results
  • Risk Factors
  • Seroepidemiologic Studies
  • Theileria / genetics
  • Theileria / isolation & purification
  • Ticks

Conflict of Interest Statement

ETHICS APPROVAL AND CONSENT TO PARTICIPATE: The collection of blood samples from horses in the state of Rio de Janeiro, Brazil was approved by the Ethics Committee on Animal Research of the Federal Rural University of Rio de Janeiro (COMEP / UFRRJ), under process number 23083.001257/2012-53 (protocol number 201/2012). Collections of blood from horses located in Canada and the United States were approved by the University of Saskatchewan Committee on Animal Care and Supply and by the NVSL/Center for Veterinary Biologics Institutional Animal Care and Use Committee, respectively. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

This article includes 61 references
  1. Mehlhorn H, Schein E. Redescription of Babesia equi Laveran, 1901 as Theileria equi Mehlhorn, Schein 1998.. Parasitol Res 1998 Jun;84(6):467-75.
    doi: 10.1007/s004360050431pubmed: 9660136google scholar: lookup
  2. Nutall GHF, Strickland C. Die parasiten der pferdepiroplasmose resp. der “biliary fever”. Zentralbl Bakt Mikrobiol 1910;1:524–525.
  3. de Waal DT. Equine piroplasmosis: a review.. Br Vet J 1992 Jan-Feb;148(1):6-14.
    doi: 10.1016/0007-1935(92)90061-5pubmed: 1551016google scholar: lookup
  4. Thompson PH. Ticks as vectors of equine piroplasmosis.. J Am Vet Med Assoc 1969 Jul 15;155(2):454-7.
    pubmed: 5816131
  5. Phipps LP, Otter A. Transplacental transmission of Theileria equi in two foals born and reared in the United Kingdom.. Vet Rec 2004 Mar 27;154(13):406-8.
    doi: 10.1136/vr.154.13.406pubmed: 15083978google scholar: lookup
  6. Scoles GA, Ueti MW. Vector ecology of equine piroplasmosis.. Annu Rev Entomol 2015 Jan 7;60:561-80.
    doi: 10.1146/annurev-ento-010814-021110pubmed: 25564746google scholar: lookup
  7. Scoles GA, Ueti MW. Amblyomma cajennense is an intrastadial biological vector of Theileria equi.. Parasit Vectors 2013 Oct 23;6(1):306.
    doi: 10.1186/1756-3305-6-306pmc: PMC4028807pubmed: 24499587google scholar: lookup
  8. Scoles GA, Hutcheson HJ, Schlater JL, Hennager SG, Pelzel AM, Knowles DP. Equine piroplasmosis associated with Amblyomma cajennense Ticks, Texas, USA.. Emerg Infect Dis 2011 Oct;17(10):1903-5.
    doi: 10.3201/eid1710.101182pmc: PMC3310643pubmed: 22000367google scholar: lookup
  9. Friedhoff KT, Tenter AM, Müller I. Haemoparasites of equines: impact on international trade of horses.. Rev Sci Tech 1990 Dec;9(4):1187-94.
    doi: 10.20506/rst.9.4.535pubmed: 2132711google scholar: lookup
  10. 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
  11. 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
  12. Grause JF, Ueti MW, Nelson JT, Knowles DP, Kappmeyer LS, Bunn TO. Efficacy of imidocarb dipropionate in eliminating Theileria equi from experimentally infected horses.. Vet J 2013 Jun;196(3):541-6.
    doi: 10.1016/j.tvjl.2012.10.025pubmed: 23199699google scholar: lookup
  13. Knowles DP Jr, Kappmeyer LS, Stiller D, Hennager SG, Perryman LE. Antibody to a recombinant merozoite protein epitope identifies horses infected with Babesia equi.. J Clin Microbiol 1992 Dec;30(12):3122-6.
    pmc: PMC270599pubmed: 1280648doi: 10.1128/jcm.30.12.3122-3126.1992google scholar: lookup
  14. Wise LN, Kappmeyer LS, Mealey RH, Knowles DP. Review of equine piroplasmosis.. J Vet Intern Med 2013 Nov-Dec;27(6):1334-46.
    doi: 10.1111/jvim.12168pubmed: 24033559google scholar: lookup
  15. Knowles DP Jr, Perryman LE, Kappmeyer LS, Hennager SG. Detection of equine antibody to Babesia equi merozoite proteins by a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.. J Clin Microbiol 1991 Sep;29(9):2056-8.
    pmc: PMC270260pubmed: 1774334doi: 10.1128/jcm.29.9.2056-2058.1991google scholar: lookup
  16. Rhalem A, Sahibi H, Lasri S, Johnson WC, Kappmeyer LS, Hamidouch A, Knowles DP, Goff WL. Validation of a competitive enzyme-linked immunosorbent assay for diagnosing Babesia equi infections of Moroccan origin and its use in determining the seroprevalence of B. equi in Morocco.. J Vet Diagn Invest 2001 May;13(3):249-51.
    doi: 10.1177/104063870101300311pubmed: 11482604google scholar: lookup
  17. Kappmeyer LS, Perryman LE, Hines SA, Baszler TV, Katz JB, Hennager SG, Knowles DP. 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 1999 Jul;37(7):2285-90.
    pmc: PMC85139pubmed: 10364599doi: 10.1128/jcm.37.7.2285-2290.1999google scholar: lookup
  18. 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
  19. Rapoport A, Aharonson-Raz K, Berlin D, Tal S, Gottlieb Y, Klement E, Steinman A. Molecular characterization of the Babesia caballi rap-1 gene and epidemiological survey in horses in Israel.. Infect Genet Evol 2014 Apr;23:115-20.
    doi: 10.1016/j.meegid.2014.01.033pubmed: 24524984google scholar: lookup
  20. Awinda PO, Mealey RH, Williams LB, Conrad PA, Packham AE, Reif KE, Grause JF, Pelzel-McCluskey AM, Chung C, Bastos RG, Kappmeyer LS, Howe DK, Ness SL, Knowles DP, Ueti MW. Serum antibodies from a subset of horses positive for Babesia caballi by competitive enzyme-linked immunosorbent assay demonstrate a protein recognition pattern that is not consistent with infection.. Clin Vaccine Immunol 2013 Nov;20(11):1752-7.
    doi: 10.1128/CVI.00479-13pmc: PMC3837787pubmed: 24049108google scholar: lookup
  21. Rüegg SR, Torgerson P, Deplazes P, Mathis A. Age-dependent dynamics of Theileria equi and Babesia caballi infections in southwest Mongolia based on IFAT and/or PCR prevalence data from domestic horses and ticks.. Parasitology 2007 Jul;134(Pt 7):939-47.
    doi: 10.1017/S0031182007002405pubmed: 17306055google scholar: lookup
  22. 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
  23. Battsetseg B, Lucero S, Xuan X, Claveria FG, Inoue N, Alhassan A, Kanno T, Igarashi I, Nagasawa H, Mikami T, Fujisaki K. Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction.. Vet Parasitol 2002 Aug 22;107(4):351-7.
    doi: 10.1016/S0304-4017(02)00131-0pubmed: 12163246google scholar: lookup
  24. Schwint ON, Knowles DP, Ueti MW, Kappmeyer LS, Scoles GA. Transmission of Babesia caballi by Dermacentor nitens (Acari: Ixodidae) is restricted to one generation in the absence of alimentary reinfection on a susceptible equine host.. J Med Entomol 2008 Nov;45(6):1152-5.
    doi: 10.1093/jmedent/45.6.1152pubmed: 19058641google scholar: lookup
  25. Nagore D, García-Sanmartín J, García-Pérez AL, Juste RA, Hurtado A. Detection and identification of equine Theileria and Babesia species by reverse line blotting: epidemiological survey and phylogenetic analysis.. Vet Parasitol 2004 Aug 13;123(1-2):41-54.
    doi: 10.1016/j.vetpar.2004.04.010pubmed: 15265570google scholar: lookup
  26. Alhassan A, Thekisoe OM, Yokoyama N, Inoue N, Motloang MY, Mbati PA, Yin H, Katayama Y, Anzai T, Sugimoto C, Igarashi I. Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis.. Vet Parasitol 2007 Jan 31;143(2):155-60.
    doi: 10.1016/j.vetpar.2006.08.014pubmed: 16973284google scholar: lookup
  27. 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
  28. 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
  29. 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
  30. 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
  31. Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JD, Wengenack NL, Rosenblatt JE, Cockerill FR 3rd, Smith TF. Real-time PCR in clinical microbiology: applications for routine laboratory testing.. Clin Microbiol Rev 2006 Jan;19(1):165-256.
    doi: 10.1128/CMR.19.1.165-256.2006pmc: PMC1360278pubmed: 16418529google scholar: lookup
  32. 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
  33. Ueti MW, Palmer GH, Kappmeyer LS, Statdfield M, Scoles GA, Knowles DP. Ability of the vector tick Boophilus microplus to acquire and transmit Babesia equi following feeding on chronically infected horses with low-level parasitemia.. J Clin Microbiol 2005 Aug;43(8):3755-9.
    doi: 10.1128/JCM.43.8.3755-3759.2005pmc: PMC1233951pubmed: 16081906google scholar: lookup
  34. Schwint ON, Ueti MW, Palmer GH, Kappmeyer LS, Hines MT, Cordes RT, Knowles DP, Scoles GA. Imidocarb dipropionate clears persistent Babesia caballi infection with elimination of transmission potential.. Antimicrob Agents Chemother 2009 Oct;53(10):4327-32.
    doi: 10.1128/AAC.00404-09pmc: PMC2764191pubmed: 19620328google scholar: lookup
  35. Ueti MW, Mealey RH, Kappmeyer LS, White SN, Kumpula-McWhirter N, Pelzel AM, Grause JF, Bunn TO, Schwartz A, Traub-Dargatz JL, Hendrickson A, Espy B, Guthrie AJ, Fowler WK, Knowles DP. Re-emergence of the apicomplexan Theileria equi in the United States: elimination of persistent infection and transmission risk.. PLoS One 2012;7(9):e44713.
    doi: 10.1371/journal.pone.0044713pmc: PMC3435266pubmed: 22970295google scholar: lookup
  36. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 1999;41:95–98.
  37. Applied Biosystems. Creating standard curves with genomic DNA or plasmid DNA templates for use in quantitative PCR. 2003.
  38. Altman DG. Practical statistics for medical research. 1. London: Chapman and Hall; 1991.
  39. Agresti A, Coull BA. Approximate is better than “exact” for interval estimation of binomial proportions. Am Stat 1998;52:119–126.
  40. Kreader CA. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein.. Appl Environ Microbiol 1996 Mar;62(3):1102-6.
    pmc: PMC167874pubmed: 8975603doi: 10.1128/aem.62.3.1102-1106.1996google scholar: lookup
  41. 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.2014.12.006pmc: PMC4356873pubmed: 25830110google scholar: lookup
  42. Kappmeyer LS, Perryman LE, Knowles DP Jr. A Babesia equi gene encodes a surface protein with homology to Theileria species.. Mol Biochem Parasitol 1993 Nov;62(1):121-4.
    doi: 10.1016/0166-6851(93)90185-Zpubmed: 8114813google scholar: lookup
  43. Kappmeyer LS, Thiagarajan M, Herndon DR, Ramsay JD, Caler E, Djikeng A, Gillespie JJ, Lau AO, Roalson EH, Silva JC, Silva MG, Suarez CE, Ueti MW, Nene VM, Mealey RH, Knowles DP, Brayton KA. Comparative genomic analysis and phylogenetic position of Theileria equi.. BMC Genomics 2012 Nov 9;13:603.
    doi: 10.1186/1471-2164-13-603pmc: PMC3505731pubmed: 23137308google scholar: lookup
  44. Criado-Fornelio A, Buling A, Asenzo G, Benitez D, Florin-Christensen M, Gonzalez-Oliva A, Henriques G, Silva M, Alongi A, Agnone A, Torina A, Madruga CR. Development of fluorogenic probe-based PCR assays for the detection and quantification of bovine piroplasmids.. Vet Parasitol 2009 Jun 10;162(3-4):200-6.
    doi: 10.1016/j.vetpar.2009.03.040pubmed: 19376655google scholar: lookup
  45. Alanazi AD, Said AE, Morin-Adeline V, Alyousif MS, Slapeta J. Quantitative PCR detection of Theileria equi using laboratory workflows to detect asymptomatic persistently infected horses.. Vet Parasitol 2014 Dec 15;206(3-4):138-45.
    doi: 10.1016/j.vetpar.2014.09.019pubmed: 25450724google scholar: lookup
  46. Pitel PH, Pronost S, Scrive T, Léon A, Richard E, Fortier G. Molecular detection of Theileria equi and Babesia caballi in the bone marrow of asymptomatic horses.. Vet Parasitol 2010 May 28;170(1-2):182-4.
    doi: 10.1016/j.vetpar.2010.01.043pubmed: 20185243google scholar: lookup
  47. Peckle M, Pires MS, Dos Santos TM, Roier EC, da Silva CB, Vilela JA, Santos HA, Massard CL. Molecular epidemiology of Theileria equi in horses and their association with possible tick vectors in the state of Rio de Janeiro, Brazil.. Parasitol Res 2013 May;112(5):2017-25.
    doi: 10.1007/s00436-013-3360-0pmc: PMC3625414pubmed: 23474658google scholar: lookup
  48. Bhoora R, Franssen L, Oosthuizen MC, Guthrie AJ, Zweygarth E, Penzhorn BL, Jongejan F, Collins NE. Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa.. Vet Parasitol 2009 Feb 5;159(2):112-20.
    doi: 10.1016/j.vetpar.2008.10.004pubmed: 19019541google scholar: lookup
  49. Liu Q, Meli ML, Zhang Y, Meili T, Stirn M, Riond B, Weibel B, Hofmann-Lehmann R. Sequence heterogeneity in the 18S rRNA gene in Theileria equi from horses presented in Switzerland.. Vet Parasitol 2016 May 15;221:24-9.
    doi: 10.1016/j.vetpar.2016.03.003pubmed: 27084467google scholar: lookup
  50. Qablan MA, Oborník M, Petrželková KJ, Sloboda M, Shudiefat MF, Hořín P, Lukeš J, Modrý D. Infections by Babesia caballi and Theileria equi in Jordanian equids: epidemiology and genetic diversity.. Parasitology 2013 Aug;140(9):1096-103.
    doi: 10.1017/S0031182013000486pubmed: 23673249google scholar: lookup
  51. Butler CM, Sloet van Oldruitenborgh-Oosterbaan MM, Stout TA, van der Kolk JH, Wollenberg Lv, Nielen M, Jongejan F, Werners AH, Houwers DJ. Prevalence of the causative agents of equine piroplasmosis in the South West of The Netherlands and the identification of two autochthonous clinical Theileria equi infections.. Vet J 2012 Aug;193(2):381-5.
    doi: 10.1016/j.tvjl.2011.12.014pubmed: 22266019google scholar: lookup
  52. Machado RZ, Toledo CZ, Teixeira MC, André MR, Freschi CR, Sampaio PH. Molecular and serological detection of Theileria equi and Babesia caballi in donkeys (Equus asinus) in Brazil.. Vet Parasitol 2012 May 25;186(3-4):461-5.
    doi: 10.1016/j.vetpar.2011.11.069pubmed: 22186194google scholar: lookup
  53. Posada-Guzmán MF, Dolz G, Romero-Zúñiga JJ, Jiménez-Rocha AE. Detection of Babesia caballi and Theileria equi in Blood from Equines from Four Indigenous Communities in Costa Rica.. Vet Med Int 2015;2015:236278.
    doi: 10.1155/2015/236278pmc: PMC4663365pubmed: 26649225google scholar: lookup
  54. Rosales R, Rangel-Rivas A, Escalona A, Jordan LS, Gonzatti MI, Aso PM, Perrone T, Silva-Iturriza A, Mijares A. Detection of Theileria equi and Babesia caballi infections in Venezuelan horses using Competitive-Inhibition ELISA and PCR.. Vet Parasitol 2013 Sep 1;196(1-2):37-43.
    doi: 10.1016/j.vetpar.2013.02.004pubmed: 23582233google scholar: lookup
  55. Mahmoud MS, Kandil OM, Nasr SM, Hendawy SH, Habeeb SM, Mabrouk DM, Silva MG, Suarez CE. Serological and molecular diagnostic surveys combined with examining hematological profiles suggests increased levels of infection and hematological response of cattle to babesiosis infections compared to native buffaloes in Egypt.. Parasit Vectors 2015 Jun 12;8:319.
    doi: 10.1186/s13071-015-0928-9pmc: PMC4467044pubmed: 26062684google scholar: lookup
  56. Romero-Salas D, Mira A, Mosqueda J, García-Vázquez Z, Hidalgo-Ruiz M, Vela NA, de León AA, Florin-Christensen M, Schnittger L. Molecular and serological detection of Babesia bovis- and Babesia bigemina-infection in bovines and water buffaloes raised jointly in an endemic field.. Vet Parasitol 2016 Feb 15;217:101-7.
    doi: 10.1016/j.vetpar.2015.12.030pubmed: 26827869google scholar: lookup
  57. Rodrigues VDS, Garcia MV, Cruz BC, Maciel WG, Zimmermann NP, Koller WW, Barros JC, Andreotti R. Life cycle and parasitic competence of Dermacentor nitens Neumann, 1897 (Acari: Ixodidae) on different animal species.. Ticks Tick Borne Dis 2017 Mar;8(3):379-384.
    doi: 10.1016/j.ttbdis.2016.12.014pubmed: 28063831google scholar: lookup
  58. Arthur DR. Ticks: a monograph of the Ixodoidea. Cambridge: Cambridge University Press; 1960.
  59. Krause PJ, Daily J, Telford SR, Vannier E, Lantos P, Spielman A. Shared features in the pathobiology of babesiosis and malaria.. Trends Parasitol 2007 Dec;23(12):605-10.
    doi: 10.1016/j.pt.2007.09.005pubmed: 17988944google scholar: lookup
  60. Doolan DL, Dobaño C, Baird JK. Acquired immunity to malaria.. Clin Microbiol Rev 2009 Jan;22(1):13-36, Table of Contents.
    doi: 10.1128/CMR.00025-08pmc: PMC2620631pubmed: 19136431google scholar: lookup
  61. Jackson AP, Otto TD, Darby A, Ramaprasad A, Xia D, Echaide IE, Farber M, Gahlot S, Gamble J, Gupta D, Gupta Y, Jackson L, Malandrin L, Malas TB, Moussa E, Nair M, Reid AJ, Sanders M, Sharma J, Tracey A, Quail MA, Weir W, Wastling JM, Hall N, Willadsen P, Lingelbach K, Shiels B, Tait A, Berriman M, Allred DR, Pain A. The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction.. Nucleic Acids Res 2014 Jun;42(11):7113-31.
    doi: 10.1093/nar/gku322pmc: PMC4066756pubmed: 24799432google scholar: lookup

Citations

This article has been cited 6 times.
  1. 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
  2. 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
  3. 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
  4. Vasconcelos VO, Costa EGL, Moreira VR, Morais-Costa F, Duarte ER. Efficacy of plants extracts from the Cerrado against adult female of Dermacentor nitens (Acari: Ixodidae). Exp Appl Acarol 2018 Aug;75(4):419-427.
    doi: 10.1007/s10493-018-0276-5pubmed: 30073431google scholar: lookup
  5. Wang T, Chen X, Yan X, Su Y, Gao W, Liu C, Wang W. Progress in serology and molecular biology of equine parasite diagnosis: sustainable control strategies. Front Vet Sci 2025;12:1663577.
    doi: 10.3389/fvets.2025.1663577pubmed: 40979365google scholar: lookup
  6. 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
Subscribe to our newsletter
Join 99,357 horse owners like you who receive our email updates on horse health and nutrition.