Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization

Abstract

A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species of Theileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to the T. sergenti-T. buffeli-T. orientalis group. The Babesia species included were Babesia bovis, B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria and Babesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences from Bos taurus or other hemoparasites (Trypanosoma species, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigemina were identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species.

FIG. 1

Locations of species-specific oligonucleotides (shaded) in the 18S rRNA V4 hypervariable region. The first T. orientalis isolate listed is type D (underlined A at position 668), and the second is the T. orientalis isolate described as T. buffeli Warwick (double-underlined T at position 668). A mix of these two oligonucleotides was used on the blot. The melting temperature (Tm) of each oligonucleotide is indicated. The ratio of each oligonucleotide to the catchall Theileria and Babesia oligonucleotide (a ratio of 1 correspond to 200 pmol) is also indicated. The catchall Theileria and Babesia oligonucleotide is identical for all 10 sequences. The R at position 871 in the T. annulata sequence denotes either an A or a G.

FIG. 2

RLB of PCR products obtained from Theileria, Babesia, and other bovine hemoparasite samples. PCR products are applied in vertical lanes. Species-specific oligonucleotides are applied in horizontal rows. Lanes 1 to 7, T. annulata isolates from Turkey (lane 1), India (lane 2), Spain (lane 3), Mauritania (lane 4), Sudan (lane 5), Portugal (lane 6), and Bahrain (lane 7); lanes 8 and 9, T. parva isolates from Tanzania and Kenya, respectively; lanes 10 and 11, T. mutans isolates from Tanzania and Nigeria, respectively; lane 12, T. taurotragi; lane 13, T. velifera; lanes 14 to 21, T. orientalis isolates from Texas (lane 14), Australia (lane 15), Japan (lane 16), England (lane 17), northwest China (lane 18), China (lane 19), Iran (lane 20), and Korea (lane 21); lane 22, B. bovis; lane 23, B. bigemina; lane 24, B. divergens; lane 25, B. major; lane 26, bovine DNA; lane 27, Trypanosoma congolense; lane 28, Trypanosoma vivax; lane 29, Trypanosoma brucei; lane 30, A. marginale; lane 31, Cowdria ruminantium; lane 32, Ehrlichia canis. Rows: 1, B. divergens; 2, B. bigemina; 3, B. bovis; 4, T. orientalis; 5, T. velifera; 6, T. taurotragi; 7, T. mutans; 8, T. parva; 9, T. annulata; 10, catchall Theileria and Babesia control oligonucleotide.

FIG. 3

Sensitivity of the PCR and the RLB assay. (A) PCR using RLB-F and RLB-R primers on DNA extracted from serial dilutions of T. annulata-infected blood. Lanes 1 and 13, molecular size markers; lanes 2 to 9, PCR products derived from DNA extracted from serial dilutions representing 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, and 10⁻⁸% parasitemia, respectively; lane 10, 0% parasitemia; lane 11, distilled water control (−); lane 12, PCR positive control of 10 ng of genomic DNA of T. annulata (+). (B) Corresponding RLB of PCR products derived from DNA extracted from serial dilutions of T. annulata-infected blood. Lanes 2 to 12 are identical to lanes 2 to 12 in panel A; lanes 1 and 13 are empty. Rows 1 and 2, T. annulata-specific and catchall Theileria and Babesia control oligonucleotides, respectively.

FIG. 4

Competition between B. bovis and T. annulata DNAs in the RLB-PCR. Lanes 1 to 7 each contain 500 fg of B. bovis DNA, as well as T. annulata DNA in the following amounts: 0 pg (lane 1), 0.1 pg (lane 2), 1.0 pg (lane 3), 10 pg (lane 4), 0.1 ng (lane 5), 1.0 ng (lane 6), and 10 ng (lane 7). Lane 8 is empty (−). Lanes 9 to 15 contain the amounts of T. annulata DNA present in lanes 1 to 7, respectively, but no B. bovis DNA. Rows are identical to those in Fig. 2.

FIG. 5

RLB of mixed DNA samples from parasites that occur in the same region. Lane 1, distilled water control (neg.); lanes 2 through 10, as labeled; lane 12, mix I, containing T. annulata, T. orientalis, B. bovis, B. bigemina, and B. divergens; lane 14, mix II, containing T. parva, T. mutans, T. taurotragi, T. velifera, and T. orientalis. Lanes 11 and 13 are empty (−). Rows are identical to those in Fig. 2.

FIG. 6

RLB with PCR products derived from DNA extracted from blood samples from animals infected with two different parasites as indicated. Lane 1, preinfection/day of T. annulata infection (marked by arrow); lane 2, 2 weeks post-T. annulata infection; lane 3, 12 weeks post-T. annulata infection/day of infection with second parasite (marked by arrow); lane 4, 1 week post-second infection; lane 5, 2 weeks post-second infection; lane 6, 3 weeks post-second infection; lane 7, 4 weeks post-second infection; lane 8, 5 weeks post-second infection; lane 9, 6 weeks post-second infection; lane 10, 7 weeks post-second infection. Rows are identical to those in Fig. 2.

FIG. 7

RLB with PCR products derived from DNA extracted from field samples collected in Spain. (A) Lanes: 1 to 8 and 10 to 29, samples from Toledo; 9, Càdiz sample; 30, PCR distilled water control (−); 31, 10 ng of T. annulata as a positive control (+). The sample positive for both T. annulata and B. bigemina is marked with an arrow. (B) Lanes: 1 to 17, samples from Càdiz; 18, empty (x); 19, distilled water control (−); 20, 10 ng of T. annulata as a positive control (+). The twice-measured Càdiz sample is marked with an asterisk in both blots. Rows are identical to those in Fig. 2.

FIG. 7

RLB with PCR products derived from DNA extracted from field samples collected in Spain. (A) Lanes: 1 to 8 and 10 to 29, samples from Toledo; 9, Càdiz sample; 30, PCR distilled water control (−); 31, 10 ng of T. annulata as a positive control (+). The sample positive for both T. annulata and B. bigemina is marked with an arrow. (B) Lanes: 1 to 17, samples from Càdiz; 18, empty (x); 19, distilled water control (−); 20, 10 ng of T. annulata as a positive control (+). The twice-measured Càdiz sample is marked with an asterisk in both blots. Rows are identical to those in Fig. 2.