First molecular detection and characterization of zoonotic Bartonella species in fleas infesting domestic animals in Tunisia

Abstract

Background: Bartonellosis is an emerging vector-borne disease caused by different intracellular bacteria of the genus Bartonella (Rhizobiales: Bartonellaceae) that is transmitted primarily by blood-sucking arthropods such as sandflies, ticks and fleas. In Tunisia, there are no data available identifying the vectors of Bartonella spp. In our research, we used molecular methods to detect and characterize Bartonella species circulating in fleas collected from domestic animals in several of the country's bioclimatic areas.

Results: A total of 2178 fleas were collected from 5 cats, 27 dogs, 34 sheep, and 41 goats at 22 sites located in Tunisia's five bioclimatic zones. The fleas were identified as: 1803 Ctenocephalides felis (83%) (Siphonaptera: Pulicidae), 266 C. canis (12%) and 109 Pulex irritans (5%) (Siphonaptera: Pulicidae). Using conventional PCR, we screened the fleas for the presence of Bartonella spp., targeting the citrate synthase gene (gltA). Bartonella DNA was detected in 14% (121/866) of the tested flea pools [estimated infection rate (EIR) per 2 specimens: 0.072, 95% confidence interval (CI): 0.060-0.086]. The Bartonella infection rate per pool was broken down as follows: 55% (65/118; EIR per 2 specimens: 0.329, 95% CI: 0.262-0.402) in C. canis; 23.5% (8/34; EIR per 2 specimens: 0.125, 95% CI: 0.055-0.233) in P. irritans and 6.7% (48/714; EIR per 2 specimens: 0.032, 95% CI: 0.025-0.045) in C. felis. Infection rates, which varied significantly by bioclimatic zone (P < 0.0001), were highest in the humid areas. By sequencing, targeting the gltA gene and the 16S-23S rRNA Intergenic Spacer Regions (ITS), we identified three Bartonella zoonotic species: B. elizabethae, B. henselae, B. clarridgeiae, as well as uncharacterized Bartonella genotypes.

Conclusions: To the best of our knowledge, this is the first time that fleas in Tunisia have been shown to carry zoonotic species of Bartonella. The dog flea, Ctenocephalides canis, should be considered the main potential vector of Bartonella. Our study not only provides new information about this vector, but also offers a public health update: medical practitioners and farmers in Tunisia should be apprised of the presence of Bartonella in fleas and implement preventive measures.

Keywords: B. clarridgeiae; B. henselae; Bartonella elizabethae; Bartonellosis; C. canis; Ctenocephalides felis; Pulex irritans; Tunisia; Zoonotic Bartonella species.

Fig. 1

Map of Tunisia showing the distribution of flea collection sites and repartition of Bartonella infected fleas according to bioclimatic areas

Fig. 2

Phylogenetic tree based on citrate synthase (gltA) gene of Bartonella spp. using the MEGA 5.02 software. The tree was obtained using the neighbor-joining method. Numbers at the nodes are proportions of 1000 bootstrap resampling that support the topology. Brucella melitensis strain Br.m-1268/11-Geo MjBr9.1 was used as the outgroup to root the tree. TUN (Tunisia) sequences detected in this work have been deposited in GenBank under the accession numbers KP126460 – KP126480. ●: Ctenocephalides felis; ■: Ctenocephalides canis; ▲: Pulex irritans