Flow cytometric evaluation of selected antimicrobial efficacy for clearance of Anaplasma marginale in short-term erythrocyte cultures.

The tick-borne rickettsia, Anaplasma marginale, causes the economically important cattle disease anaplasmosis. Once infected, cattle remain lifelong carriers. Herein, we used flow cytometry to test the efficacy of three antimicrobials; oxytetracycline, imidocarb and enrofloxacin against Virginia (VGN) or Oklahoma (OK) A. marginale isolates in short-term erythrocyte cultures. Parasite viability was assessed using the vital dye hydroethidine (HE), which is detectable when living organisms convert HE to ethidium bromide. Viability of A. marginale in selected cultures was determined by subinoculation into susceptible calves. Data were analyzed by MANOVA, Tukey-Kramer honest significant difference and Wilcoxon rank sum tests. Receiver operating characteristic (ROC) analysis was used to correlate results with culture infectivity. Enrofloxacin inhibited A. marginale in a dose dependent manner. Surprisingly, higher concentrations of imidocarb were less effective than lower concentrations against A. marginale with significant differences (P < 0.05) observed between the two isolates. Oxytetracycline was the least active drug tested. Cultures infected with the OK isolate exposed to 4.0 microg/mL enrofloxacin and those of the VGN and OK isolates exposed to 1.0 microg/mL imidocarb were sterilized. This is the first in vitro study demonstrating the efficacy of enrofloxacin against A. marginale. Furthermore, these data indicate that flow cytometry is a useful assay for screening antimicrobials against A. marginale.