DETECTION OF ANAPLASMA-MARGINALE DNA IN BOVINE ERYTHROCYTES BY SLOT-BLOT AND IN-SITU HYBRIDIZATION WITH A PCR-MEDIATED DIGOXIGENIN-LABELED DNA-PROBE

A 409-base pair (bp) DNA fragment derived from the msp-1 beta gene of Anaplasma marginale was amplified and simultaneously labeled with digo xigenin-11-dUTP by a polymerase chain reaction (PCR) assay. The result ing digoxigenin-labeled 409-bp PCR product was used as a probe for slo t-blot and in situ hybridization to detect A. marginale DNA from exper imentally infected bovine erythrocytes. The hybrid formation was detec ted with alkaline phosphatase-conjugated anti-digoxigenin antibody and substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazo lium salt. In slot-blot hybridizations, the probe detected A. marginal e DNA from approximately 1,000-10,000 infected erythrocytes in 1.25 mi of whole blood, which is equivalent to a parasitemia level of 0.0000 1%. The probe proved to be A. marginale-specific when tested with 17 s pecies of microorganisms. The applicability of the probe for diagnosis was tested by screening A. marginale infections in 2 experimentally i nfected splenectomized cattle before microscopically detectable parasi temias and after acute infection. After inoculation of infected blood, A. marginale infections were detected with the probe 14 days prior to detection in stained smears. Microscopically inapparent parasitemias were also detected with the probe for 2 months after acute disease. Wh en the probe was used for in situ hybridization on methanol-fixed bloo d smears, probe reaction could be visualized with light microscopy on A. marginale inclusions within infected erythrocytes. The probe reacti on was not observed on leukocytes and uninfected erythrocytes from inf ected blood smears, on erythrocytes from uninfected blood samples, or on samples infected with A. ovis, Babesia bovis, or B. bigemina. This PCR-mediated nonradioactive probe appears to be a sensitive diagnostic test for A. marginale.