Bartonella rochalimae and Bartonella vinsonii subsp. berkhoffii co-infection in Pulex irritans from dogs, Italy

Background: Bartonella spp. are important emerging vector-borne pathogens, worldwide. Today, the Bartonella genus consists of at least 33 species and three subspecies. They are fastidious, hemotropic, Gram-negative bacteria, mainly transmitted by bloodsucking arthropods and/or animal scratches. Fleas play a major role in the natural cycle of different Bartonella spp. In this study, Bartonella spp. were detected and genetically characterized from fleas infesting a human patient as well as dogs and livestock living in close contact with him, in a farm in southern Italy. Material and methods: The study was performed in a farm in Ostuni (Brindisi, Apulia region, Italy), in animals managed according to a traditional semi-intensive system. Fleas were collected from a 35 year-old man, living in the farm in two small rooms located close to animal pens. The patient was affected by severe itchy, skin reactions and heavy flea infestation seeking for medical care. All animals present in the farm were clinically observed and flea infestation was recorded. Sheep, dogs and a cat, appeared anemic, with restlessness and weakness, and presented patchy alopecia and skin hardness mainly in limbs. Fleas (n=69) were collected from a German shepherd (n=19) and a Maremmano sheepdog (n=13), from two sheep (n=5), a cat (n=1) and from the human patient (n=31). Fleas were morphologically identified at species level and screened for Bartonella spp. by conventional PCRs targeting RNA polymerase B (rpoB), citrate synthase (gltA), 60 kDa heat shock protein (groEL), cell division protein (ftsZ) gene fragments, Bartonella 16S–23S internal transcribed spacer (ITS) and sequence analysis. Results: All fleas were identified as Pulex irritans (n=23 males, n=46 females). Bartonella DNA was detected in six out of 32 fleas (18.8%; 95% CI: 5.2-32.3), both male (n=2) and female (n=4), from two dogs. Based on the gltA/groEL/rpoB/ftsZ/ITS analyses the identified Bartonella genotypes were highly similar or identical to strains previously detected in dog or other host species from different parts of the world. gltA/groEL/rpoB/ftsZ/ITS fragments, 100% identical to Bartonella vinsonii subsp. berkhoffii were detected in four fleas (three females and one male) from the German shepherd. Another male flea from the German shepherd carried DNA of both Bartonella vinsonii subsp. berkhoffii and Bartonella rochalimae. One female flea from the Maremmano sheepdog carried gltA/groEL/rpoB/ftsZ/ITS fragments that were closely related to Bartonella rochalimae (>99% similarity). Conclusion: This report supports the role of Pulex irritans as potential vector of Bartonella spp. including B. vinsoni subsp. berkhoffii and B. rochalimae, the latter was firstly detected in fleas from Italy. Our findings are consistent with those of other studies that reported the detection of the zoonotic B. rochalimae from arthropods, including fleas and ticks. In this study, all Bartonella spp. strains were detected in P. irritans fleas biting dogs, thus further suggesting the role of dogs as reservoirs of zoonotic Bartonella spp. Indeed, B. vinsoni subsp. berkhoffii causes bacteremia and endocarditis in dogs and has been implicated as an agent of a blood culture-negative endocarditis in a human. Keywords: Bartonella rochalimae, Bartonella vinsonii subsp. berkhoffii, fleas, Pulex irritans, dog, Italy.