ABSTRACTThe presence ofBartonellaspp. was investigated in domestic ungulates grazing in communal pastures from a mountain area in northern Spain, where 18.3% (17/93) of cattle were found to be positive by PCR combined with a reverse line blot (PCR/RLB), whereas sheep (n= 133) or horses (n= 91) were found not to be infected by this pathogen.Bartonellainfection was significantly associated with age, since older animals showed a higher prevalence than heifers and calves. In contrast to other studies,B. chomeliiwas the most frequent species found in cattle (14/17), whileB. boviswas detected in only three animals. Moreover, 18B. chomeliiisolates and oneB. bovisisolate were obtained from nine animals. Afterwards,B. chomeliiisolates were characterized by a multilocus sequence typing (MLST) method which was adapted in this study. This method presented a high discrimination power, identifying nine different sequence types (STs). This characterization also showed the presence of different STs simultaneously in the same host and that STs had switched over time in one of the animals. In addition,B. chomeliiSTs seem to group phylogenetically in two different lineages. The onlyB. bovisisolate was characterized with a previously described MLST method. This isolate corresponded to a new ST which is located in lineage I, where theB. bovisstrains infectingBos taurussubsp.taurusare grouped. Further studies on the dynamics ofBartonellainfection in cattle and the potential ectoparasites involved in the transmission of this microorganism should be performed, improving knowledge about the interaction ofBartonellaspp. and domestic ungulates.