Anaplasma marginale: detection of carrier cattle by PCR-ELISA

A highly sensitive and specific polymerase chain reaction (PCR) based assay for the detection of the minute levels of Anaplasma marginale present in the blood of long-term carrier cattle was developed. A simple lysis method was used to remove most of the haemoglobin from the blood to facilitate direct input of samples into the PCR reactions without prior purification of the DNA. PCR product was detected by enzyme-linked immunosorbent assay (ELISA) to simplify the processing of large numbers of samples. The sensitivity limit of the PCR-ELISA was 0.00015% parasitaemia (24 infected erythrocytes per microlitre of blood). No cross-reactivity of the assay was observed when A. marginale-negative blood infected with Babesia bovis or Theileria orientalis was tested. The PCR-ELISA was shown to be 92% efficient in the detection of long-term A. marginale carrier cattle. No false-positive results were obtained. These results compared favourably with 2 serological assays for detection of A. marginale carrier cattle (card agglutination test and ELISA) which were applied to the same experimental animals.