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The main objective of this study was to determine the prevalence of ' Bartonella' spp. and hemoplasma infection in healthy pet cats in Ontario. Blood samples collected from July 2006 to March 2007, were tested for ' Bartonella' spp. by PCR (646 samples) and by blood culture (896 samples, April 2006 to March 2007), and by PCR for diagnosis of hemoplasma (742 samples). The overall prevalence of 'Bartonella' spp. and hemoplasma was 4.3% (3.7% 'Bartonella henselae,' 0.6% 'Bartonella clarridgeiae') and 4% (3.3% 'Mycoplasma hemominutum,' 0.7% 'Mycoplasma hemofelis'), repectively. The presence of ' M. turicensis,' was identified by PCR amplification and sequencing, for the first time in North America. The other goal of the study was to investigate the presence of ' Rickettsia felis' in cat fleas in Ontario. A pool of one to five fleas from 50 infested cats were collected and used for PCR. The presence of ' R. felis' was confirmed in 9 of 50 samples.