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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.

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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