In-vitro studies on the susceptibility of ovine strains of Anaplasma phagocytophilum to antimicrobial agents and to immune serum

Abstract

Five strains of Anaplasma phagocytophilum, the causative agent of tick-borne fever of sheep and cattle and human granulocytic anaplasmosis, were tested in vitro for their susceptibility to nine antibiotics using the continuous tick cell line ISE6. Minimum inhibitory concentrations (MICs) were evaluated by comparing the percentage of infection of Giemsa-stained antibiotic-treated Anaplasma phagocytophilum-infected cells with that of untreated controls after 6 days of culture. The minimum bactericidal concentrations (MBCs) were evaluated after washing infected cells with antibiotic-free medium and further incubation of 6 days before comparing the percentage of infection of Giemsa-stained antibiotic-treated and untreated cells and by comparing the number of copies of the p44 gene by real-time polymerase chain reaction using p44-specific primers. The tick cell culture system was also used to assay the possible neutralizing effects of immune serum on cell-free bacteria in vitro. The neutralizing effects of immune serum were evaluated by comparing the number of copies of the p44 gene in samples inoculated with cell-free bacteria after 1h incubation with two-fold dilutions of immune sera obtained 21 days after infection with those inoculated with cell-free bacteria after incubation for 1h with two-fold dilutions of sera obtained before infection. Doxycycline, rifampin and ciprofloxacin were the most effective compounds, with MICs of 0.125 microg/ml, 0.5 mg/ml and 1 microg/ml, respectively. There was total resistance to penicillin, ampicillin, ceftriaxone and streptomycin and only very limited susceptibility to gentamycin and chloramphenicol. Both doxycycline and rifampin were also bactericidal at the same concentrations. Exposure of bacteria to immune ovine sera resulted in significant reductions of the number of copies of p44 gene.