[Evaluation of sensitivity of PCR for detecting DNA of Trypanosoma vivax with several methods of blood sample preparations].
Parasitological techniques show a low sensitivity for diagnosis of active infections with Trypanosoma sp. in livestock, particularly in the case of chronic infections. T. vivax antigen detection through antigen-ELISA developed by Nantulya and Lindqvist (1989) is not sensitive and specific enough for infection diagnosis. T. vivax DNA detection through polymerase chain reaction (PCR) using the oligonucleotides developed by MASIGA et coll. (1992) appears to be an alternative for a specific diagnosis of T. vivax active infections in livestock. Twenty-two blood samples containing known numbers of T. vivax/ml, ranging from 1 to 1767, were prepared by dilution of T. vivax infected sheep blood into blood from a non infected sheep. PCR sensitivity was evaluated in several types of blood sample preparations: crude heparinized blood, plasma, lysed blood, buffy coat from haematocrit capillary tubes, pellet from plasma centrifugation, and DNA purified with an ion exchange resin commercial kit. Crude heparinized blood almost always inhibited PCR. Sensitivity of PCR with plasma and lysed blood was low, around 450 parasites/ml. PCR on buffy coat was more sensitive, but PCR products were sometimes little visible. Pellet of plasma centrifugation is an original, fast and economic preparation, whose PCR products are highly visible and which presents a high sensitivity: one hundred percent of the samples were positive when the parasitaemia was over 9 parasites/ml. DNA purification is slightly more time consuming and expensive, since it requires several manipulations and the use of a commercial kit, but it appears to be the most sensitive technique among those investigated: one hundred percent of the samples were positive when the parasitaemia was over 2 parasites/ml; however, PCR products were sometimes difficult to interpret. These last two techniques are recommended for a sensitive and species-specific diagnosis of active infections of livestock with T. vivax. These techniques should be evaluated for other pathogenic trypanosome species of livestock.