Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa
Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa; Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa; Utrecht Centre for Tick-borne Diseases (UCTD), Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Yalelaan 1, 3584 CL Utrecht, Netherlands; Department of Equine Sciences, Medicine Section, Utrecht University, PO Box 80.153, 3508 TD Utrecht, Netherlands; Agricultural Research Council - Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110, South Africa
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. © 2009 Elsevier B.V. All rights reserved.
protozoal DNA; RNA 18S; analytic method; animal experiment; article; Babesia caballi; blotting; controlled study; equine piroplasmosis; erythrocyte; fluorescent antibody technique; gene identification; genetic variability; genotype; horse disease; nonhuman; nucleotide sequence; quantitative study; real time polymerase chain reaction; reverse line blot; RNA sequence; sensitivity analysis; South Africa; Theileria; Theileria equi; theileriosis; Animals; Babesia; Babesiosis; DNA, Protozoan; Genotype; Horse Diseases; Horses; Male; Polymerase Chain Reaction; Reproducibility of Results; RNA, Ribosomal, 18S; Sensitivity and Specificity; South Africa; Theileria; Theileriasis; Babesia; Babesia caballi; Equidae; Theileria equi