Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction.
Abstract: We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.
Publication Date: 2001-10-06 PubMed ID: 11587829DOI: 10.1016/s0304-4017(01)00471-xGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research involves the use of a specific type of polymerase chain reaction (PCR) to detect the presence of a parasite, Babesia equi, in horse blood cells. This technique allows for the detection of very small amounts of the parasite and helps identify variations in the parasite’s genetic makeup.
Methodology
- The researchers use a nested polymerase chain reaction (PCR), a sensitive technique that allows for the detection and amplification of a specific DNA sequence.
- The PCR was specifically designed to amplify a 102bp DNA fragment of the Babesia equi parasite, based on the sequence of a known merozoite antigen gene (ema-1).
- To test the specificity of the PCR, researchers tried to amplify DNA from other parasites including Babesia caballi, Babesia bovis, and Babesia bigemina which didn’t provide positive results, confirming the specificity for B. equi.
Testing
- The technique’s sensitivity was tested in a mock infection, where the researchers found that only six infected cells in a sample of 10^8 equine blood cells were needed for reliable detection of the parasite.
- This corresponds to a parasitemia (parasite infection in the blood) level of only 0.000006%, indicating the impressive sensitivity of this method.
Genetic Variations
- In addition to detecting the presence of the parasite, the researchers also found genetic variation in the B. equi samples they tested.
- They did this by using another technique, PCR-RFLP (PCR combined with restriction fragment length polymorphism), which allows for the identification of variations in the DNA sequences of the 102bp fragment and the entire ema-1 gene.
- This genetic variation was confirmed by sequencing the entire ema-1 gene from one of the B. equi isolates.
Practical Applications
- Based on the ability to sensitively and specifically detect B. equi, along with the ability to identify genetic variations, the researchers suggest that this nested PCR technique is valuable for routine detection of this parasite in horses, especially those chronically infected.
- Further, it also has potential uses in epidemiological and phylogenetic studies, to track the spread and evolution of the parasite, and in monitoring the effect of chemotherapeutic trial treatments on horses infected with B. equi.
Cite This Article
APA
Nicolaiewsky TB, Richter MF, Lunge VR, Cunha CW, Delagostin O, Ikuta N, Fonseca AS, da Silva SS, Ozaki LS.
(2001).
Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction.
Vet Parasitol, 101(1), 9-21.
https://doi.org/10.1016/s0304-4017(01)00471-x Publication
Researcher Affiliations
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, 91501-970 Porto Alegre, RS, Brazil.
MeSH Terms
- Amino Acid Sequence
- Animals
- Antigens, Protozoan
- Babesia / genetics
- Babesia / isolation & purification
- Babesiosis / diagnosis
- Babesiosis / veterinary
- Base Sequence
- Chronic Disease
- DNA, Protozoan / analysis
- Erythrocytes / parasitology
- Gene Amplification
- Horse Diseases / diagnosis
- Horses
- Membrane Proteins / genetics
- Molecular Sequence Data
- Molecular Weight
- Polymerase Chain Reaction / veterinary
- Polymorphism, Restriction Fragment Length
- Protozoan Proteins / genetics
- Restriction Mapping / veterinary
- Sensitivity and Specificity
- Sequence Analysis, DNA / veterinary
Citations
This article has been cited 16 times.- Yang G, Zhou B, Chen K, Hu Z, Guo W, Wang X, Du C. Diagnostic Performance of Competitive ELISA and Western Blot Methods for the Detection of Antibodies against Theileria equi and Babesia caballi.. Microorganisms 2022 Dec 21;11(1).
- Mongruel ACB, Medici EP, da Costa Canena A, Calchi AC, Perles L, Rodrigues BCB, Soares JF, Machado RZ, André MR. Theileria terrestris nov. sp.: A Novel Theileria in Lowland Tapirs (Tapirus terrestris) from Two Different Biomes in Brazil.. Microorganisms 2022 Nov 23;10(12).
- 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.
- 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.
- Yang G, Chen K, Guo W, Hu Z, Qi T, Liu D, Wang Y, Du C, Wang X. Development of a Test Card Based on Colloidal Gold Immunochromatographic Strips for Rapid Detection of Antibodies against Theileria equi and Babesia caballi.. Microbiol Spectr 2022 Feb 23;10(1):e0241121.
- 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.
- 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).
- 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.
- 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.
- 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.
- 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.
- Hines SA, Ramsay JD, Kappmeyer LS, Lau AO, Ojo KK, Van Voorhis WC, Knowles DP, Mealey RH. Theileria equi isolates vary in susceptibility to imidocarb dipropionate but demonstrate uniform in vitro susceptibility to a bumped kinase inhibitor.. Parasit Vectors 2015 Jan 20;8:33.
- 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.
- 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.
- 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.
- 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.
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