First Molecular Detection and Characterization of Zoonotic
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Zouari et al. Parasites & Vectors (2017) 10:436 DOI 10.1186/s13071-017-2372-5 RESEARCH Open Access First molecular detection and characterization of zoonotic Bartonella species in fleas infesting domestic animals in Tunisia Saba Zouari, Fatma Khrouf, Youmna M’ghirbi and Ali Bouattour* 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’sbioclimaticareas. 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,aswellas 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.Thedogflea,Ctenocephalides canis,shouldbeconsideredthemainpotentialvectorofBartonella. Our study not only provides new information about this vector, but also offers a public health update: medical practitioners andfarmersinTunisiashouldbeapprisedofthepresenceofBartonella in fleas and implement preventive measures. Keywords: Bartonella elizabethae, B. henselae, B. clarridgeiae, Ctenocephalides felis, C. canis, Pulex irritans, Bartonellosis, Zoonotic Bartonella species, Tunisia * Correspondence: [email protected]; [email protected] Université de Tunis El Manar, Institut Pasteur de Tunis, Laboratoire d’Epidémiologie et de Microbiologie Vétérinaire LR11IPT03, Service d’Entomologie Médicale, 1002 Tunis-Belvédère, Tunisie © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zouari et al. Parasites & Vectors (2017) 10:436 Page 2 of 9 Background sheep) in 22 sites located in Tunisia’s five bioclimatic Global changes, including climate and land use, are zones (humid, sub-humid, semi-arid, arid and Saharan, among the most significant influences on the distribution Fig. 1). Individual fleas were placed in labeled flasks and and density of hematophagous arthropod species, which stored in 70% ethanol. All specimens were identified to may affect zoonotic diseases including bartonellosis, species level using the standard taxonomic keys [14]. A caused by bacteria of the genus Bartonella (fastidious distribution map of collected fleas, according to the bio- hemotropic Gram-negative, facultative intracellular organ- climatic areas, was made using ArcGIS 9 software (ver- isms) belonging to the α2-subgroup of the proteobacteria. sion 9.3.1). These organisms can infect animal and human erythro- cytes and endothelial cells and cause polymorphic clinical DNA extraction from fleas diseases [1]. A host can be infected by Bartonella by trans- For DNA extraction, fleas from the same species, of the fusions/organ transplants, arthropod saliva (fleas, sand same sex, from the same host in the same site were flies, lice and ticks) or by flea feces in the case of Cat- pooled (2 fleas per pool, to reduce testing costs). The Scratch-Disease (CSD) [1, 2]. identified fleas were removed from the 70% ethanol Several tools that are useful and more sensitive than flasks, washed, dried, and minced with disposable scal- culture and serological techniques, including conven- pels, and placed in 200 μl ATL buffer. We followed the tional PCR, RFLP, MST, MLVA and also the pre- manufacturer’s instructions for the QIAamp® DNA tissue enrichment culture using Bartonella-Alpha-Proteobac- extraction kit (Qiagen, Hilden, Germany). The final elu- teria-Growth Medium combined with PCR and sequen- tion step was done using 90 μl of AE buffer and then cing (ePCR) [3], have been developed to simultaneously stored at -20 °C for further use. To monitor for contam- detect and characterize Bartonella species and subspe- ination during the processes, an extraction control (dis- cies in hosts and vectors [4, 5]. More than 35 species tilled water) for every 10 flea pools was added in each and three subspecies have been identified to date, of extraction experiment. The DNA concentration was de- which 17 species are involved or associated with an termined with a NanoDrop spectrophotometer (Nano- expanding spectrum of animal and human diseases. Drop ND-1000 Technologies, Wilmington, DE), and These include Carrion’s disease, trench fever, bacillary ranged between 50 and 400 ng/μl. For the PCR reaction, angiomatosis, endocarditis and CSD [6, 7]. CSD the the extracted DNA was diluted to reach a suitable con- most common zoonotic infection caused by Bartonella centration (50 ng/μl). henselae, which is transmitted by cat fleas, Ctenocepha- lides felis. Bartonella henselae, which is frequently trans- Detection of Bartonella spp. by PCR, DNA sequencing and mitted to humans by a cutaneous trauma (scratch and/ phylogenetic relationships or bite) from an infected cat [8], was recently implicated Fleas were screened for the presence of Bartonella spp. in an intraocular inflammation caused by CSD in using the citrate synthase gene (gltA). The amplification Tunisia [9]. Several cases of B. quintana endocarditis of Bartonella DNA was carried out as described by were also reported [10, 11]. Norman et al. [15] using the forward primer BhCS.7p Serological studies conducted in Tunisia showed ca- (5′-GGG GAC CAG CTC ATG GTG G-3′) and the re- nine seropositivity for B. vinsonii, B. henselae, B. clarrid- verse primer BhCS.1137n (5′-AAT GCA AAA AGA geiae and B. bovis. This correlated well with tick and/or ACA GTA AAC A-3′), yielding a fragment of approxi- flea infestations of the dogs that were tested [12]. More- mately 379 bp. over, a recent molecular study detected “Candidatus B. PCR amplifications were performed in a final volume of merieuxii” DNA in dogs [13]. While several clinical, mo- 25 μl containing 2.5 μl of 10× Taq buffer (Invitrogen, CA, lecular and serological studies have demonstrated that USA), 1,5 μl of MgCl2 (1.5 mM), 2.5 μlofdeoxyribonucle- Bartonella species circulate among patients and dogs in otide triphosphate (200 μM), 0.5 μl of each primer Tunisia [8–10, 12], no data about the role of fleas as po- (200 nM) and 0.75 U of Taq Platinum polymerase (Invi- tential vectors are available. trogen, CA, USA), followed by the addition of 3 μlofflea This research used molecular methods to detect and DNA extracts. The amplification reaction was carried out characterize Bartonella species circulating in fleas col- in a thermocycler (Perkin Elmer 2400). PCR cycling con- lected from domestic animals and to compare its infec- ditions were 10 min at 94 °C for an initial denaturation, tion rates in Tunisia’s various bioclimatic areas. followed by 35 cycles of 30 s at 94 °C, 45 s at 51 °C, and 30 s at 72 °C, with a final extension of 7 min at 72 °C. Methods Positive and negative controls were included in each series Flea collection and identification of experiments. To avoid false-positive results, negative An entomological investigation was carried out to collect controls included sterile water and Bartonella-negative fleas infesting domestic animals (dogs, cats, goats and fleas DNA were added as the templates in each run. DNA Zouari et al. Parasites & Vectors (2017) 10:436 Page 3 of 9 Fig. 1 Map of Tunisia showing the distribution of flea collection sites and repartition of Bartonella infected fleas according to bioclimatic areas extracted from B. elizabethae culture (kindly provided by rRNA Intergenic Spacer Regions (ITS) used the primers Pr. Zanzen, Hospital Hedi Chaker Sfax, Tunisia) was used ITS2F (5′-GGG GCC GTA GCT CAG CTG-3′)and as a positive control. To avoid cross-contamination, the ITS2R (5′-TGA ATA TAT CTT CTC TTC ACA ATT extraction, amplification and gel electrophoresis were TC-3′) [16]. For the ITS region, the PCR cycling condi- done in separate rooms. PCR products were visualized by tions were performed as follows: 15 min at 94 °C for an ethidium bromide staining after electrophoresis in a 1.5% initial denaturation followed by 35 cycles of 1 min at 94 ° agarose gel (Gellyphor, EuroClone, Milan, Italy) and their C, 35 s at 60 °C, and 30 s at 72 °C, with a final extension size was estimated by comparing them with Gene of 7 min at 72 °C.