Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa.
Abstract: A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB) probe was developed for the detection of Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The B. caballi TaqMan MGB qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized erythrocytes (PE). We further evaluated a previously reported Theileria equi-specific qPCR assay and showed that it was able to detect the 12 T. equi 18S rRNA sequence variants previously identified in South Africa. Both qPCR assays were tested on samples from two ponies experimentally infected with either T. equi or B. caballi. The qPCR assays were more sensitive than the indirect fluorescent antibody test (IFAT) and the reverse-line blot (RLB) during the early onset of the disease. The assays were subsequently tested on field samples collected from 41 horses, resident on three stud farms in the Northern Cape Province, South Africa. The IFAT detected circulating T. equi and B. caballi antibody in, respectively, 83% and 70% of the samples. The RLB detected T. equi parasite DNA in 73% of the samples, but none of the samples were positive for B. caballi, although 19 T. equi-positive samples also hybridized to the Babesia genus-specific probe. This could indicate a mixed T. equi and B. caballi infection in these samples, with either the B. caballi parasitaemia at a level below the detection limit of the B. caballi RLB probe, or the occurrence of a novel Babesia genotype or species. In contrast, the qPCR assays correlated fairly well with the IFAT. The B. caballi TaqMan MGB qPCR assay was able to detect B. caballi parasite DNA in 78% of the samples. The T. equi-specific qPCR assay could positively detect T. equi DNA in 80% of the samples. These results suggest that the qPCR assays are more sensitive than the RLB assay for the detection of T. equi and B. caballi infections in field samples.
Publication Date: 2009-11-20 PubMed ID: 20031328DOI: 10.1016/j.vetpar.2009.11.011Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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Research in South Africa sought to develop and evaluate real-time PCR assays for detecting diseases in horses specifically caused by Babesia caballi and Theileria equi. It was found that these assays were efficient, specific, and often more sensitive than other testing methods.
Development of Real-time PCR Assay
- The researchers developed a real-time polymerase chain reaction (qPCR) assay with the aim to detect Babesia caballi infection in horses. Babesia caballi is a parasite that infects the red blood cells of animals, particularly horses, causing a disease known as babesiosis.
- To design the primers and probes essential for the qPCR assay, the team used nine previously published sequences situated in the V4 hypervariable region of the B. caballi 18S rRNA gene. The aim was to locate unique and conserved regions of the gene.
- The resulting qPCR assay proved to be both efficient and specific in detecting B. caballi. The detection limit, or the minimum concentration at which positive samples could be reliably detected, was 0.000114% of parasitized red blood cells.
Assay Testing and Comparisons
- The team then tested the previously reported Theileria equi-specific qPCR assay, another parasite that causes a similar disease in horses. This assay successfully detected the 12 T. equi 18S rRNA gene variants previously identified in South Africa.
- Two ponies infected with either T. equi or B. caballi were subjected to both these qPCR assays. The assays demonstrated more sensitivity during the early onset of the disease compared to two other detection methods: the indirect fluorescent antibody test (IFAT) and the reverse line blot (RLB).
Field Testing and Results
- The researchers conducted field testing on samples collected from 41 horses on three stud farms in the Northern Cape Province of South Africa.
- The IFAT was able to detect antibodies of T. equi and B. caballi in 83% and 70% of the samples respectively. The RLB, on the other hand, confirmed the presence of T. equi parasite DNA in 73% of the samples, but failed to detect B. caballi in any.
- However, the qPCR assays performed notably well. The B. caballi qPCR assay managed to detect B. caballi DNA in 78% of the samples, and the T. equi-specific qPCR assay was able to detect T. equi DNA in 80% of the samples.
Conclusion
- These results suggest that the qPCR assays developed and tested in this research study offer a more sensitive method for detecting T. equi and B. caballi infections in field samples compared to the RLB assay.
Cite This Article
APA
Bhoora R, Quan M, Franssen L, Butler CM, van der Kolk JH, Guthrie AJ, Zweygarth E, Jongejan F, Collins NE.
(2009).
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, 168(3-4), 201-211.
https://doi.org/10.1016/j.vetpar.2009.11.011 Publication
Researcher Affiliations
- Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, South Africa. raksha.bhoora@up.ac.za
MeSH Terms
- Animals
- Babesia / genetics
- Babesiosis / diagnosis
- Babesiosis / veterinary
- DNA, Protozoan / analysis
- Genotype
- Horse Diseases / diagnosis
- Horse Diseases / parasitology
- Horses
- Male
- Polymerase Chain Reaction
- RNA, Ribosomal, 18S / genetics
- Reproducibility of Results
- Sensitivity and Specificity
- South Africa
- Theileria / genetics
- Theileriasis / diagnosis
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