TY - JOUR
T1 - Specific detection and identification of African trypanosomes in bovine peripheral blood by means of a PCR-ELISA assay
AU - Cabrera, Leyda
AU - De Witte, Jacob
AU - Victor, Björn
AU - Vermeiren, Lieve
AU - Zimic, Mirko
AU - Brandt, Jef
AU - Geysen, Dirk
PY - 2009/10/14
Y1 - 2009/10/14
N2 - The aim of the present study was to develop a PCR-ELISA assay for the detection and differentiation of the main African pathogen trypanosomal species present in peripheral blood of cattle. The proposed methodology allows to specifically differentiate Trypanosoma congolense, Trypanosoma vivax and the subgenus Trypanozoon, by means of a sensitive universal PCR amplifying trypanosome DNA followed by an ELISA-based hybridization with three highly specific probes. The semi-nested PCR had a sensitivity of 15 fg, 15 fg, and 0.15 fg of DNA from T. vivax, T. congolense, and Trypanosoma brucei brucei, respectively that is sufficient to detect parasites in blood during the chronic phase of the disease. Biotinylated second round asymmetric PCR amplification products were used in an ELISA set up using three species-specific probes for the diagnosis of T. congolense (type Riverine, Kilifi or Savannah), T. vivax and T. brucei brucei. A factor O.D. of 0.082 was determined on blood samples from bovines (n = 18) from a non-endemic area in Africa. In a pilot study of blood samples of naturally and experimentally Trypanosoma infected cattle previously characterized by PCR-RFLP (n = 42), a high rate of concordance (93.3%) was found between PCR-RFLP and PCR-ELISA. There is a good ratio between positive and negative O.D. values (3.00 vs. 0.1) and the technique can also be used to distinguish mixed infections.
AB - The aim of the present study was to develop a PCR-ELISA assay for the detection and differentiation of the main African pathogen trypanosomal species present in peripheral blood of cattle. The proposed methodology allows to specifically differentiate Trypanosoma congolense, Trypanosoma vivax and the subgenus Trypanozoon, by means of a sensitive universal PCR amplifying trypanosome DNA followed by an ELISA-based hybridization with three highly specific probes. The semi-nested PCR had a sensitivity of 15 fg, 15 fg, and 0.15 fg of DNA from T. vivax, T. congolense, and Trypanosoma brucei brucei, respectively that is sufficient to detect parasites in blood during the chronic phase of the disease. Biotinylated second round asymmetric PCR amplification products were used in an ELISA set up using three species-specific probes for the diagnosis of T. congolense (type Riverine, Kilifi or Savannah), T. vivax and T. brucei brucei. A factor O.D. of 0.082 was determined on blood samples from bovines (n = 18) from a non-endemic area in Africa. In a pilot study of blood samples of naturally and experimentally Trypanosoma infected cattle previously characterized by PCR-RFLP (n = 42), a high rate of concordance (93.3%) was found between PCR-RFLP and PCR-ELISA. There is a good ratio between positive and negative O.D. values (3.00 vs. 0.1) and the technique can also be used to distinguish mixed infections.
KW - African bovine trypanosomes
KW - Diagnosis
KW - PCR-ELISA
KW - T. brucei
KW - T. congolense
KW - T. vivax
KW - Trypanozoon
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U2 - 10.1016/j.vetpar.2009.06.017
DO - 10.1016/j.vetpar.2009.06.017
M3 - Article
C2 - 19619947
AN - SCOPUS:70349165075
SN - 0304-4017
VL - 164
SP - 111
EP - 117
JO - Veterinary Parasitology
JF - Veterinary Parasitology
IS - 2-4
ER -