Leptospirosis is a zoonotic infection with a worldwide distribution that is associated with both endemic disease and epidemics, with the incidence of disease being highest in tropical climates [17]. The number of cases of leptospirosis diagnosed has increased since 1983 in North America [36]. Lyme disease is a multisystemic disorder caused by infection with Borrelia burgdorferi and is the most common tick-associated illness in North America [4,7,8].

Leptospirosis and Lyme disease have some overlapping geographic distributions and both can produce acute febrile disease. Although they may be self-limiting diseases in some cases, they can also have significant morbidity and mortality if not prevented or treated. The lack of rapid and accurate diagnostic testing of leptospirosis and Lyme disease often dictate empirical therapy for acute febrile illness. As the differential diagnosis of an acute febrile illness in any particular setting may be extensive, therapeutics that may cover a broad range of infectious diseases are desirable. Doxycycline and penicillin are currently the drugs of choice in the treatment of human leptospirosis, based chiefly on the fact that they are the only agents that have been studied in randomized controlled clinical trials [12,21,29]. Previous studies have shown that the newer cephalosporins and other β-lactams, as well as fluoroquinolone antibiotics, all have good in vitro activity against strains of Leptospira [26,34]. Doxycyline and tetracycline have also been used in the treatment of equine and canine borreliosis [10,35].

Although many in vitro and in vivo studies have been performed to find an optimal treatment for spirochetal disease, preferred forms of treatment are still not quite clear. Moreover, the majority of studies concerning in vitro susceptibility testing of leptospires and borreliae against antimicrobial agents were conducted with organisms recovered from human infections. The antimicrobial susceptibility of leptospires and borreliae isolated from animals is not well known. In this investigation, in vitro antimicrobial susceptibility tests were conducted with 6 spirochetal strains (4 leptospires and 2 borreliae) isolated from animal clinical cases against 3 antimicrobial agents (amoxicillin, tilmicosin, and enrofloxacin), which are used in the treatment of animal diseases.

A uniform and standardized method to test antimicrobial susceptibility against pathogenic spirochetes has not been established. For borreliae and leptospires, the MIC is generally considered the drug concentration at which no motile organisms are observed by dark-field microscopy after 48-72 h of incubation [2,18]. The MBCs for leptospires and borreliae are determined by the absence of spirochetes in subcultures on microscopic examination after various incubation periods [18]. The MBC determinations made by these methods require virtually 100% killing of the final inoculum, which is strict requirement for any antimicrobial agent [9]. In this study, the MBC of antibiotics for leptospires and borreliae was defined as the lowest concentration of the antibiotics at which no spirochetes were subcultured. The macrolide antibiotic tilmicosin produced excellent in vitro activity against all 6 strains of spirochetes tested. The MIC and MBC of amoxicillin against Leptospira spp. were higher than those of tilmicosin and enrofloxacin, and the MBC of amoxicillin against Leptospira spp. was higher than noted MICs. However, enrofloxacin was not effective against B. burgdorferi tested, as shown by its high MIC and MBC.

Tilmicosin is a novel long-acting macrolide antibiotic developed exclusively for veterinary use [28]. As Mycotil, it is used to treat bovine respiratory disease associated with pasteurellae [16,23]. Tilmicosin (10 mg/kg, once) and amoxicillin (15 mg/ml, once or twice) were effective in cattle for resolving experimental leptospirosis caused by L. borgpetersenii serovar Hardjo [3,32]. Enrofloxacin was used concurrently with ampicillin in canine leptospirosis and 83% of infected dogs survived [1]. Amoxicillin is effective in treatment of canine Lyme disease [15]. However, there are no data on the clinical use of enrofloxacin or tilmicosin in borreliosis of animals.

Based upon our findings, tilmicosin was superior in vitro on a microgram-per-milliliter basis when tested alongside amoxicillin and enrofloxacin under identical test conditions in EMJH or BSK-II media. Moreover, maximum concentrations of tilmicosin in plasma after a subcutaneous dose of 10 mg/kg to cattle are over 100 times higher than the MIC against borreliae and tilmicosin has high volume of distribution that selectively accumulates in solid tissues [22,38]. The accumulation and concentration of tilmicosin in tissues could be due to its lipophilic properties, which favor passage through the lipid membrane of the cells. One mechanism by which the diffusion of tilmicosin may have led to enhance intracellular accumulation is the tendency of basic compounds to be lysosomotropic and become trapped in lysosomes as a result of the acidic pH [30]. The Lyme borrelia reside in the acidic endosome and the use of a lysosomotropic agent augments the clinical activity of macrolide antibiotics in treatment of human patients with chronic borreliosis [11]. Therefore, the potential role of tilmicosin in the treatment of leptospirosis and Lyme disease in some animal species merits further evaluation. Although no significant adverse effects have been reported in cattle, injections of Mycotil to horses, goats, swine, or nonhuman primates can be fatal. Extensive studies on its safety in dogs have not been performed. The heart is the target of toxicity in animals, perhaps mediated via depletion of cardiac intracellular calcium, resulting in a negative inotropic effect [20]. Other formulations of the tilmicosin would be necessary before it could be used in these species.

Classical macrolides and azalides frequently fail in the therapy of early Lyme disease in human and clinical relapse has been observed following conclusion of treatment [14,19, 27,37]. Moreover, it has been speculated that resistance may develop in borreliae preexposed to erythromycin owing to resistant subpopulations. We have evaluated antibiotic treatment using tetracycline, doxycline, and ceftiofur in experimental borreliosis of dogs and horses, however, most of them can not completely eliminate the persistent infection [6,33]. The lack of effective agents in the treatment of animal borreliosis made it urgent to identify other potential antibiotics. Therefore, further evaluation of the potential use of a non-toxic formulation of tilmicosin in the treatment of equine or canine leptopsirosis and/or borreliosis should be confirmed.