Fleas of the genus Ctenocephalides are the most common ectoparasites infesting dogs and cats world-wide. The species Ctenocephalides felis and Ctenocephalides canis are competent vectors for zoonotic pathogens such as Rickettsia felis and Bartonella spp. Improved knowledge on the diversity and phylogenetics of fleas is important for understanding flea-borne pathogen transmission cycles. Fleas infesting privately owned dogs and cats from the Czech Republic (n=97) and Romania (n=66) were subjected to morphological and molecular identification and phylogenetic analysis. There were a total of 59 (60.82%) cat fleas (Ctenocephalides felis felis), 30 (30.93%) dog fleas (Ctenocephalides canis), 7 (7.22%) European chicken fleas (Ceratophyllus gallinae) and 1 (1.03%) northern rat flea (Nosopsyllus fasciatus) collected in the Czech Republic. Both C. canis and C. felis felis were identified in Romania. Mitochondrial DNA sequencing at the cox1 gene on a cohort of 40 fleas revealed the cosmopolitan C. felis felis clade represented by cox1 haplotype 1 is present in the Czech Republic. A new C. felis felis clade from both the Czech Republic and Romania is also reported. A high proportion of C. canis was observed from dogs and cats in the current study and phylogeny revealed that C. canis forms a sister clade to the oriental cat flea Ctenocephalides orientis (syn. C. felis orientis). Out of 33 fleas tested, representing C. felis felis, C. canis and Ce. gallinae, 7 (21.2%) were positive for R. felis using diagnostic real-time PCR targeting the gltA gene and a conventional PCR targeting the ompB gene. No samples tested positive for Bartonella spp. using a diagnostic real-time PCR assay targeting ssrA gene. This study confirms high genetic diversity of C. felis felis globally and serves as a foundation to understand the implication for zoonotic disease carriage and transmission by the flea genus Ctenocephalides.

Department of Medical Entomology University of Sydney and Pathology West ICPMR Westmead Hospital Westmead New South Wales 2145 Australia

Department of Parasitology and Parasitic Diseases University of Agricultural Sciences and Veterinary Medicine Cluj Napoca Calea Mănăştur 3 5 400372 Cluj Napoca Romania

Department of Pathological Morphology and Parasitology Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Palackého 612 42 Brno Czech Republic

Department of Pathological Morphology and Parasitology Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Palackého 612 42 Brno Czech Republic; Central European Institute of Technology University of Veterinary and Pharmaceutical Sciences Brno Czech Republic; Biology Centre Institute of Parasitology Academy of Sciences of the Czech Republic Ceske Budejovice Czech Republic

Dermatology Unit Clinics of Small Animals and Horses University of Veterinary Medicine Vienna Austria

Faculty of Veterinary and Agricultural Sciences The University of Melbourne Parkville Victoria 3052 Australia

Faculty of Veterinary Science McMaster Building B14 The University of Sydney New South Wales 2006 Australia

Faculty of Veterinary Science McMaster Building B14 The University of Sydney New South Wales 2006 Australia; Department of Medical Entomology University of Sydney and Pathology West ICPMR Westmead Hospital Westmead New South Wales 2145 Australia

School of Veterinary Science The University of Queensland Gatton Queensland 4343 Australia; Faculty of Veterinary and Agricultural Sciences The University of Melbourne Parkville Victoria 3052 Australia

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