Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting
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Characteristic sequence signatures were identified within the hypervariable region 4 (V4 region) of the small ribosomal RNA gene of ovine/caprine piroplasm species including Theileria lestoquardi, T. ovis , T. separata , Babesia ovis , B. motasi , B. crassa [comprising strains B. crassa (Iran) and B. crassa (Turkey)] and several novel species: Theileria sp. 1 (China), Theileria sp. 2 (China) and Babesia sp. (China), [comprising strain Babesia sp. (Lintan), and Babesia sp. (Ningxian)] as defined previously. Based on the ascertained gene variations a reverse line blotting (RLB) assay was developed enabling direct, concurrent, highly specific and sensitive identification of virtually all presently known ovine/caprine piroplasm species. All probes bound to their respective target sequence only, therefore, no cross-reaction was observed resulting in clear recognition of either individual strains, species or groups. No signal was observed when ovine and caprine genomic DNA was used as the control, demonstrating that the signals are due to the presence of parasite DNA in investigated samples. Furthermore, the sensitivity of RLB could be considerably enhanced to detect a parasitemia level of at least 10-12% by reamplification of PCR products (nested PCR) thereby substantially increasing the possibility of identifying carrier animals.
KeywordsSmall Ruminant Babesia Reverse Line Blotting Babesia Species Specific Oligonucleotide Probe
This work was supported by the following European Union INCO-DEV Program contracts: contract no. ICA4-CT2000–300028 (Molecular and immunological characterisation of merozoite antigens and their encoding genes of a Theileria species highly pathogenic for small ruminants in China: application for the development of diagnostics and vaccine); contract no. ICB1CT-2000–80004 ( Theileria China antigens: application for the development of diagnostics and vaccines for small ruminants in China); contract no. ICA4-CT-2000–30006 [International Consortium on Ticks and Tick-Borne Diseases (ICTTS-2)]; a bursary was awarded to Y. H.
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