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Background There is limited clinical or epidemiological knowledge regarding  Bartonella  infection in cats, and no serological studies have compared the presence of antibodies against different  Bartonella  species. Moreover, there are limited feline  Bartonella  studies investigating co-infections with other vector-borne pathogens and the associated risk factors. Therefore, the objective of this study was to investigate  Bartonella  spp. infections and co-infections with other pathogens in cats from Barcelona (Spain) based on serological and/or molecular techniques and to determine associated risk factors. Methods We studied colony and owned cats ( n  = 135). Sera were tested for  Bartonella henselae -,  Bartonella quintana -, and  Bartonella koehlerae -specific antibodies using endpoint in-house immunofluorescence antibody assays.  Bartonella  real-time PCR (qPCR) and conventional PCR (cPCR) were performed. In addition, cPCR followed by DNA sequencing was performed for other pathogenic organisms ( Anaplasma ,  Babesia ,  Cytauxzoon ,  Ehrlichia ,  Hepatozoon , hemotropic  Mycoplasma , and  Theileria  spp.). Results From 135 cats studied, 80.7% were seroreactive against at least one  Bartonella  species.  Bartonella quintana ,  B. koehlerae , and  B. henselae  seroreactivity was 67.4, 77.0, and 80.7%, respectively. Substantial to almost perfect serological agreement was found between the three  Bartonella  species. Colony cats were more likely to be  Bartonella  spp.-seroreactive than owned cats. Moreover, cats aged ≤ 2 years were more likely to be  Bartonella  spp.-seroreactive.  Bartonella  spp. DNA was detected in the blood of 11.9% ( n  = 16) of cats. Cats were infected with  B. henselae  ( n  = 12),  B. clarridgeiae  ( n  = 3), and  B. koehlerae  ( n  = 1).  Mycoplasma  spp. DNA was amplified from 14% ( n  = 19) of cat blood specimens. Cats were infected with  Mycoplasma haemofelis  ( n  = 8),  Candidatus  M. haemominutum ( n  = 6),  Candidatus  Mycoplasma turicensis ( n  = 4), and  Mycoplasma wenyonii  ( n  = 1).  Anaplasma ,  Babesia ,  Cytauxzoon ,  Ehrlichia  spp.,  Hepatozoon , and  Theileria  spp. DNA was not amplified from any blood sample. Of the 16  Bartonella  spp.-infected cats based on PCR results, six (37%) were co-infected with  Mycoplasma  spp. Conclusions Bartonella  spp. and hemoplasma infections are prevalent in cats from the Barcelona area, whereas infection with  Anaplasma  spp.,  Babesia ,  Cytauxzoon ,  Ehrlichia  spp.,  Hepatozoon , and  Theileria  infections were not detected. Co-infection with hemotropic  Mycoplasma  appears to be common in  Bartonella -infected cats. To our knowledge, this study is the first to document  M. wenyonii  is infection in cats. Graphical Abstract

Prospective serological and molecular cross-sectional study focusing on Bartonella and other blood-borne organisms in cats from Catalonia (Spain)