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 ofTheileria 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 theT. sergenti-T. buffeli-T. orientalis group. TheBabesia 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 andBabesia 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 fromBos taurus or other hemoparasites (Trypanosomaspecies, 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. bigeminawere 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.

Coinfection of Sheep withAnaplasma,TheileriaandBabesiaSpecies in the Kurdistan Region, Iraq

“…Reverse-line blotting was performed according to Gubbels et al (1999) and Schnittger et al (2004). Probes used for the detection of A. ovis and Ehrlichia ovis were 5′-ATGTGAGGATTTTATCTTTGTA and 5′-GGCTTTTGCC TCTGTGT, respectively.…”

Observed Prevalence of Tick‐borne Pathogens in Domestic Animals in Sicily, Italy during 2003–2005

“…Reverse line blot (RLB) was used for detection of Babesia/ Theileria spp. as previously described (Gubbels et al, 1999;Georges et al, 2001;Nagore et al, 2004;Schnittger et al, 2004).…”

Integrated Molecular Diagnosis of Theileria and Babesia Species of Cattle in Italy

“…Samples were then heated at 100 ° C for 10 minutes and cooled rapidly on ice and processed as described elsewhere. 8 Membranes were incubated with a conjugate labeled with a peroxidase conjugated streptavidin. A chemiluminescent reaction was performed by using ECL reagents and reactions were visualized by using ECL hyperfilm films (Amersham, UK).…”