Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonella koehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats

Domestic cats are the natural reservoir of Bartonella henselae , B . clarridgeiae and B . koehlerae . To determine the role of wild felids in the epidemiology of Bartonella infections, blood was collected from 14 free-ranging California mountain lions ( Puma concolor ) and 19 bobcats ( Lynx rufus ). Bartonella spp. were isolated from four (29%) mountain lions and seven (37%) bobcats. These isolates were characterized using growth characteristics, biochemical reactions, molecular techniques, including PCR-RFLP of selected genes or interspacer region, pulsed-field gel electrophoresis (PFGE), partial sequencing of several genes, and DNA-DNA hybridization. Two isolates were identical to B . henselae genotype II. All other isolates were distinguished from B . henselae and B . koehlerae by PCR-RFLP of the gltA gene using endonucleases Hha I, Taq I and Aci I, with the latter two discriminating between the mountain lion and the bobcat isolates. These two novel isolates displayed specific PFGE profiles distinct from B . henselae , B . koehlerae and B . clarridgeiae . Sequences of amplified gene fragments from the three mountain lion and six bobcat isolates were closely related to, but distinct from, B . henselae and B . koehlerae . Finally, DNA-DNA hybridization studies demonstrated that the mountain lion and bobcat strains are most closely related to B . koehlerae . We propose naming the mountain lion isolates B . koehlerae subsp. boulouisii subsp. nov. (type strain: L-42-94), and the bobcat isolates B . koehlerae subsp. bothieri subsp. nov. (type strain: L-17-96), and to emend B . koehlerae as B . koehlerae subsp. koehlerae . The mode of transmission and the zoonotic potential of these new Bartonell a subspecies remain to be determined.