Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction.
We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.
Nicolaiewsky
T B
TB
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, 91501-970 Porto Alegre, RS, Brazil.
Richter
M F
MF
Lunge
V R
VR
Cunha
C W
CW
Delagostin
O
O
Ikuta
N
N
Fonseca
A S
AS
da Silva
S S
SS
Ozaki
L S
LS
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan; D001403:Babesia / Q000235:genetics / Q000302:isolation & purification*; D001404:Babesiosis / Q000175:diagnosis / Q000662:veterinary*; D001483:Base Sequence; D002908:Chronic Disease; D016054:DNA, Protozoan / Q000032:analysis*; D004912:Erythrocytes / Q000469:parasitology; D005784:Gene Amplification; D006734:Horse Diseases / Q000175:diagnosis*; D006736:Horses; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012150:Polymorphism, Restriction Fragment Length; D015800:Protozoan Proteins / Q000235:genetics; D015183:Restriction Mapping / Q000662:veterinary; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000662:veterinary
D000953:Antigens, Protozoan; D016054:DNA, Protozoan; C085964:EMA-1 protein, Babesia equi; D008565:Membrane Proteins; D015800:Protozoan Proteins