Ultrastructural and fluorochromatic changes of Anaplasma marginale exposed to oxytetracycline, imidocarb and enrofloxacin in short-term erythrocyte cultures

Anaplasma marginale causes mild to severe hemoparasitic disease resulting in significant morbidity and mortality in cattle worldwide. In the absence of universally efficacious vaccines, antimicrobial therapy combined with biocontainment and biosecurity strategies are critical to control anaplasmosis. Herein, we compared the effect of oxytetracycline, imidocarb and enrofloxacin on A. marginale isolates in short-term erythrocyte cultures. Electron micrographs detailing antimicrobial-induced changes in rickettsial morphology were scored (0–4) based on ultrastructural changes. These were compared to fluorochromatic changes detected by flow cytometry (FACS) using conversion of hydroethidine (HE) to ethidium bromide (EB) by living organisms to assess viability. A. marginale infectivity in selected cultures was confirmed by subinoculation into susceptible calves. Morphology scores were analyzed using Chi-squared tests and compared to FACS data by ANOVA with isolate, drug and concentration as co-variates in the model. Only the Virginia and Oklahoma isolates exposed to 1.0 μg/ml imidocarb and the Oklahoma isolate exposed to 4.0 μg/ml enrofloxacin were sterilized following antimicrobial exposure. Rickettsia with morphology scores of 0 had significantly more EB positive cells than inclusions with morphology scores of 4 (p = 0.039). There was also a significant association between ultrastructural changes and infectivity (p = 0.0047). Furthermore, the percent EB positive cells in the antimicrobial exposed cultures was highly predictive of the probability of infectivity (p = 0.0026). This is the first study describing ultrastructural changes in A. marginale following exposure to enrofloxacin and imidocarb. These findings demonstrate that FACS and electron microscopy are useful tools to screen new antimicrobials for the use in anaplasmosis chemotherapy.