A comparison of six primer sets for detection of Trypanosoma evansi by polymerase chain reaction in rodents and Thai livestock.
To face the worldwide threat of Surra caused by Trypanosoma evansi, international organizations have stressed the need to evaluate and standardize diagnostic tools. PCR detection of T. evansi has known a great expansion during the last 20 years, but primer sets are often insufficiently assessed and compared. In this work, we compared the performances of six primer pairs-TBR1/2 (Masiga et al., 1992), ESAG6/7 (Holland et al., 2001a, b), TEPAN1/2 (Panyim et al., 1993), pMUTEC F/R (Wuyts et al., 1994), TRYP1 R/S (Desquesnes et al., 2001) and TRYP4 R/S (Desquesnes et al., unpublished)-tested with purified T. evansi DNA serial dilutions, T. evansi-infected rat blood serial dilutions and Thai dairy cattle samples. TBR1/2 primer set was able to detect 0.01 pg of purified DNA, and a parasitaemia below one parasite per ml in rat blood. They presented the highest sensitivity in cattle samples as well as a high specificity, without non-specific products nor false positive reactions out of 84 negative cattle samples tested. ESAG6/7 showed equivalent results with purified DNA and rat samples but presented non-specific products with Thai dairy cattle samples, leading to non interpretable results. TEPAN1/2 was not able to detect less than 0.1 pg of purified DNA or 50 trypanosomes/ml in rat blood. In cattle, TEPAN1/2 primers detected only 36% of the positives detected by TBR1/2. Given the parasitemic level detected, pMUTEC F/R, TRYP1 R/S and TRYP4 R/S were not more sensitive than classical microscopic examination of the buffy coat. TBR1/2, TEPAN1/2, pMUTEC F/R and TRYP4 R/S did not cross-reacted with Babesia sp., Trypanosoma theileri and Anaplasma marginale. TBR1/2 was the most sensitive primer set to detect T. evansi in purified DNA, rodent blood and cattle blood, and did not show cross reaction with the other pathogens tested: it should be therefore preferred for epidemiological surveys. These results confirmed that TBR1/2 primers remain the reference for the detection of Trypanozoon DNA and should therefore be included in subsequent evaluations of new diagnosis tools based on DNA detection.
Pruvot
Mathieu
M
Centre de Coopération internationale en Recherche Agronomique pour le Développement (CIRAD)-Département Systèmes Biologiques (Bios), UMR177-Trypanosomes, Montpellier, F-34000 France. mpruvot@ucalgary.ca
Kamyingkird
Ketsarin
K
Desquesnes
Marc
M
Sarataphan
Nachai
N
Jittapalapong
Sathaporn
S
D000818:Animals; D002417:Cattle; D017931:DNA Primers; D003612:Dairying; D005260:Female; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013785:Thailand / Q000453:epidemiology; D014345:Trypanosoma / Q000145:classification* / Q000302:isolation & purification*; D014352:Trypanosomiasis / Q000175:diagnosis / Q000453:epidemiology / Q000469:parasitology / Q000662:veterinary*
D017931:DNA Primers