The putative role of biting flies in Bartonella transmission among ruminants was investigated. Amplification of the Bartonella citrate synthase gene from 83 Hippoboscidae was detected in 94% of 48 adult Lipoptena cervi flies, 71% of 17 adult Hippobosca equina flies, 100% of 20 adult Melophagus ovinus flies, and 100% of 10 M. ovinus pupae. Our findings suggest that Hippoboscidae play a role in the transmission of Bartonella among ruminants. The vertical transmission of Bartonella in M. ovinus and the presence of Bartonella DNA in all samples suggest a symbiotic association between Bartonella and M. ovinus.Bartonella spp. are intracellular small gram-negative bacteria transmitted by blood-sucking arthropods and considered to be emerging pathogens in humans and animals (1,8,9,22). In recent years, these organisms have been identified in a wide range of wild and domestic mammals (4, 6, 7, 11), some of which have been associated with zoonoses. Recently, four new Bartonella species have been isolated from ruminants: B. schoenbuchensis and B. capreoli were recovered from wild roe deer (Capreolus capreolus) (3, 10), whereas B. bovis (3) and B. chomelii (20) were recovered from domestic cattle. There are no pathological outcomes associated with Bartonella infection in ruminants (5, 7).Arthropod vectors involved in the transmission of Bartonella spp. among ruminants are still unknown. As blood-sucking ectoparasites of ruminants, flies of the family Hippoboscidae are good candidates for the transmission of Bartonella. Among Hippoboscidae, Lipoptena, Hippobosca, and Melophagus are the three main genera which parasitize mammals (14, 18). The deer ked (Lipoptena cervi), the predominant Lipoptena species in Europe, parasitizes cervids (15), whereas the louse fly (Hippobosca equina) parasitizes cows and horses and the sheep ked (Melophagus ovinus) is a permanent ectoparasite of sheep (Ovis aries) (18).The aim of this study was to determine if Hippoboscidae could be putative vectors of Bartonella spp. in ruminants. We investigated whether Bartonella DNA could be detected in adult and pupal stages of Hippoboscidae collected from ruminants (domestic cattle and roe deer) known to be naturally infected with Bartonella spp. We extended the study to Hippoboscidae collected from sheep and horses for which no evidence of Bartonella infection had ever been demonstrated.Collection and identification of Hippoboscidae. Eighty-three Hippoboscidae flies of different species, stages, and genders were collected and taxonomically identified under a binocular lens (18). Samples were obtained from the hosts or from our parasitology collection (Table 1). Each sample was stored in absolute ethanol.DNA extraction and PCR. Each fly and pupa were washed three times in sterile water baths and once in a 70% ethanol bath and then dried. DNA was then extracted after the flies were crushed with a bead beater as previously described (16).A 380-bp fragment of the citrate synthase (gltA) gene of the genus Bartonella was amplified in fly DNA extracts by PCR, us...
-Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA extraction protocols based on an enzymatic digestion by proteinase K followed by DNA extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA extraction efficiency using method 3 is significantly higher than DNA extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency. tick / DNA extraction / PCR / nymphal stage
-Ticks are known vectors for a wide range of pathogenic microorganisms. Their role in the transmission of some others is so far only suspected. Ticks can transmit multiple pathogens, however, little is known about the co-existence of these pathogens within questing ticks. We looked for the presence of DNA from three micro-organisms, Bartonella sp., Borrelia burgdorferi sensu lato and Babesia sp. which are known or suspected tick-borne pathogens, using a cohort of 92 questing Ixodes ricinus ticks collected from pastures in northern France. DNA was extracted from each individual tick and the presence of the three pathogens was investigated using Polymerase Chain Reaction (PCR) amplification. Nine among 92 samples (9.8%) demonstrated PCR products using Bartonella specific primers, 3 among 92 (3.3%) using Borrelia burgdorferi sensu lato specific primers and 19 among 92 (20.6%) using Babesia specific primers. Seven among 92 samples (7.6%) were PCR positive for at least two of the pathogens and one sample was positive for all three. Adult ticks (12/18; 67%) showed significantly higher infection rates compared to nymphs (11/74; 15%) for all three pathogens (P < 0.001). This study is the demonstration of the simultaneous presence of Bartonella sp., Borrelia burgdorferi sensu lato and Babesia sp. in questing Ixodes ricinus ticks. questing ticks / co-infection / PCR detection
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