A tick cell line as a powerful tool to screen the antimicrobial susceptibility of the tick-borne pathogen Anaplasma marginale

Background Anaplasma marginale is the causative agent of the severe bovine anaplasmosis. The tick Rhipicephalus microplus is one of the main vectors of A. marginale in tropical and subtropical regions of world. After the tick bite, the bacterium invades and proliferates within the bovine erythrocytes, causing anemia and impairing milk production and weight gain. In addition, infection can cause abortion and high mortality in areas of enzootic instability. The immunization with live and inactivated vaccines are employed to the control acute bovine anaplasmosis. However, they do not prevent persistent infection. Therefore, infected animals, even if immunized, are reservoirs of the bacterium and contribute to the dissemination of the disease. Antimicrobials are also largely employed for the prophylaxis of bovine anaplasmosis. However, they are often used in subtlethal doses, what can select pre-existing resistant bacteria and induce genetic or phenotypic variations. Therefore, the standardization of an in vitro assay to evaluate the susceptibility of A. marginale strains to different antimicrobials is important to allow the prescription of the more effective treatment, preventing both the selection and spread of resistant strains. Results Initially the antimicrobial susceptibility of two field isolates of A. marginale (Jaboticabal and Palmeira) infecting bovines was evaluated. The least susceptible strain (Jaboticabal) was used for the standardization of an antimicrobial assay using a culture of Ixodes scapularis-derived tick cell line, ISE6. Results showed that enrofloxacin (ENRO) at 0.25, 1 or 4 μg/mL and oxytetracycline (OTC) at 4 or 16 μg/mL are the most efficient treatments, followed by OTC at 1 μg/mL and imidocarb dipropionate (IMD) at 1 or 4 μg/mL. Conclusion In the current study, we present a new in vitro assay using a tick cell line to evaluate the susceptibility of A. marginale to antimicrobials. The maintenance of such culture is much easier than the maintenance of bovine erythrocyte

). In addition, males seem to play an important role as vectors, as they perform multiple blood meals, even in more than one host, and also exhibit greater mobility and longevity compared to females (   7   ,   8 ). Transmission may also occur mechanically through either the bite of hematophagous insects (flies, mosquitoes, tabanids), which carry residues of contaminated blood on their mouthparts, or fomites, such as needles and gloves ( 2 ).
Immunization with live and inactivated vaccines has been used to control bovine anaplasmosis. However, despite both types of vaccines induce protective immunity that reduces and prevents the clinical disease, they do not prevent persistent infection. Therefore, even if immunized, infected animals are reservoirs of the bacterium and contribute to the dissemination of the disease ( 9 ). In Brazil, as in other countries, a strain of A. marginale subspecies centrale has been used as vaccine, conferring cross-immunity against A. marginale (   9   ,   10 ). Nonetheless, there are significant differences in the efficacy of this vaccine depending on the region and the isolate of the bacterium. In addition, immunization with alive A. marginale subsp. centrale causes an infection that can lead to adverse clinical reactions (   11   ,   12 ).
In addition to vaccines, antimicrobials are largely employed for the chemoprophylaxis of bovine anaplasmosis. In endemic areas in the USA, the addition of chlortetracycline to the animals' food is frequent. On the other hand, in Brazil, where the administration of antimicrobials within the animals' diet is rare, the use of subtherapeutic dosage of injectable antimicrobials, such as imidocarb dipropionate (IMD) and oxytetracycline (OTC), has become a common practice for the prevention of anaplasmosis (

13-15
). Currently, the drug of choice for the treatment of bovine anaplasmosis is OTC, but other drugs may be employed, such as IMD and enrofloxacin (ENRO) ( ). Two multidrug resistance pumps were identified in the genome of A. marginale ( 19 ). Although the clinical significance of these genes has yet to be elucidated and, to date, there are no reports on A. marginale antimicrobial resistance, the misuse of antimicrobials may favor the selection of resistant strains. Indeed, conflicting data on the success of antimicrobial therapies in endemic areas of bovine anaplasmosis suggest that distinct isolates of A. marginale exhibit differences in antimicrobial susceptibility ( 16 , 20 ). Therefore, the standardization of in vitro assays to assess the susceptibility of A. marginale strains to different antimicrobials is important to allow the prescription of the most effective treatment, preventing the spread of resistant strains and helping in the control of bovine anaplasmosis.
In the current study, we present the evaluation of the antimicrobial susceptibility of two field isolates of A. marginale (Jaboticabal and Palmeira) infecting bovines. The least susceptible strain (Jaboticabal) was used for the standardization of an antimicrobial assay using a culture of I. scapularis-derived tick cell line, ISE6. Results showed that the in vitro assay using tick cells is a practical and powerful tool to evaluate the susceptibility of A. marginale to antimicrobials.

Treatment of A. marginale infection in cattle
Considering the previously reported data on the differences in the susceptibility of  Table 1, Fig. 1). In relation to IMD treatment, the animals infected with the Jaboticabal strain were cured with two (J3 = 44 days) or three (J4 = 52 days) doses (Table 1, Fig. 1). On the other hand, the animals infected with Palmeira strain needed only a single dose to be cured (P3 and P4 = 29 days); ( Table 1, Fig. 1). Together, the results show that the Jaboticabal strain is less susceptible to both antimicrobials than the Palmeira strain. Establishment of an in vitro assay for the screening of A. marginale susceptibility to antimicrobials The Jaboticabal strain of A. marginale, which was less susceptible to the treatment with antimicrobials in vivo, was selected to establish an in vitro antimicrobial assay. To that end, we first evaluated the susceptibility of the embryonic cells of the tick I. scapularis marginale within ISE6 cells treated with either ENRO or OTC was approximately two orders of magnitude lower than the control group (free antimicrobial; FA). In fact, significant differences between the control group and either ENRO or OTC groups were observed since the fifth day post-infection. On the other hand, we did not detect any significant differences in bacterial growth in cells treated with ENRO and OTC (Fig. 3). In addition, there was a slight but significant reduction in bacterial load in the group treated with IMD compared to the control group after 5 and 7 days post-infection.
We then analyzed the bacterial growth under lower concentrations of ENRO (0.25 μg/mL, 1 μg/mL) and OTC (1 μg/mL, 4 μg/mL) than those used in the first experiment. As reference, the same concentrations used in the first experiment were employed (ENRO; 4 μg/mL and OTC; 16 μg/mL). In addition, a higher (4 μg/mL) and a lower (0.25 μg/mL) concentrations of IMD than the previously tested (1 μg/mL) were also evaluated. ENRO effectively reduced bacterial growth at all concentrations, with a bacterial load of approximately two orders of magnitude lower than the control (Fig. 4). The same effect was observed for the concentrations of 4 μg/mL and 16 μg/mL of OTC, while a minor but still significant activity was detected with 1 μg/mL. A slight but significant reduction of the bacterial load was detected with IMD at 4 μg/mL and 1 μg/mL. Conversely, we did not detect a significant difference between the IMD treatment at 0.25 μg/mL and the control group. In summary, the in vitro assays revealed that the Jaboticabal strain of A. marginale is more susceptible to ENRO and OTC than to IMD.   The antimicrobial treatment was initiated when the animal presented a

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