Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting.
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.
Schnittger
Leonhard
L
Department of Immunology and Cell Biology, Research Centre Borstel, Parkallee 22, 23845 Borstel, Germany.
Yin
Hong
H
Qi
Bai
B
Gubbels
Marc J
MJ
Beyer
Doreen
D
Niemann
Stefan
S
Jongejan
Frans
F
Ahmed
Jabbar S
JS
D000818:Animals; D001403:Babesia / Q000145:classification* / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000235:genetics; D004195:Disease Models, Animal; D014644:Genetic Variation; D005843:Geography; D006041:Goats; D008969:Molecular Sequence Data; D018512:Parasitemia / Q000175:diagnosis; D016133:Polymerase Chain Reaction; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012418:Ruminants / Q000469:parasitology*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep; D016794:Theileria / Q000145:classification* / Q000235:genetics
D017931:DNA Primers; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S