Migratory birds, ticks, and Bartonella.

Bartonella spp. infections are considered to be vector-borne zoonoses; ticks are suspected vectors of bartonellae. Migratory birds can disperse ticks infected with zoonotic pathogens such as Rickettsia and tick-borne encephalitis virus and possibly also Bartonella. Thus, in the present study 386 tick specimens collected in spring 2009 from migratory birds on the Mediterranean islands Capri and Antikythera were screened for Bartonella spp. RNA. One or more ticks were found on 2.7% of the birds. Most ticks were Hyalomma rufipes nymphs and larvae with mean infestation rates of 1.7 nymphs and 0.6 larvae per infested bird. Bartonella spp. RNA was not detected in any of the tick specimens.

Bartonella infections are widespread in wild and domesticated mammals and several new species have been described during the last decades. Thirteen Bartonella species and subspecies have, so far, been associated with human disease, e.g. catch-scratch disease, Carrión's disease, and trench fever (1). The bartonelloses are considered to be vector-borne zoonoses and for some Bartonella species the vector is known: B. henselae – fleas, B. quintana – human louse, and B. bacilliformis – phlebotomine sandflies (2). Bartonella spp. have been detected – based almost exclusively on polymerase chain reaction (PCR) – in several tick species including Ixodes ricinus, I. scapularis, I. persulcatus, Dermacentor reticulatus, Rhipicephalus sanguineus, and Carios kelleyi (2). Although Cotté et al. (3) experimentally demonstrated that I. ricinus can transmit B. henselae during a blood meal, replicating bacteria have rarely been reported in ticks and there is little support for the hypothesis that ticks are natural competent vectors of Bartonella bacteria (1).

There is a growing body of evidence showing that migratory birds are important in the dispersal of ticks infected with important human pathogens, e.g. tick-borne encephalitis virus and Rickettsia spp. (4, 5). Possibly this may also be true for Bartonella spp.

In order to investigate the potential presence of Bartonella spp. in ticks infesting birds, a total of 386 ticks was removed from migratory birds net-captured on two Mediterranean islands and later screened for the presence of Bartonella spp. RNA.

Materials and methods

A total of 7,453 springtime migratory birds were captured in mist nets at Capri bird observatory in Italy (n=4,924) and at Antikythera bird observatory in Greece (n=2,529) between April 2nd 2009 and May 18th 2009. Each captured bird was identified to species and the ears, throat, nape, and abdomen of each bird were checked for ticks. Any tick observed was removed with forceps and individually submerged in Eppendorf tubes filled with RNAlater buffer (QIAGEN) and frozen at −20°C.

The dorsal and ventral areas of each tick were photographed with a DinoLite USB-microscope. The pictures were carefully analyzed in order to determine stage and species of the tick. The morphological species identification of immature ticks is difficult, especially with regard to the genus Hyalomma (6). Therefore, to confirm the identifications based on tick morphology a molecular approach was chosen for 10 larval and nymphal tick specimens, identified morphologically as Hyalomma sp. and considered to be representative for the whole Hyalomma sample. Available sequences of the different genes of Hyalomma species were compared in the GeneBank, and the mitochondrial 12S rDNA was identified as an appropriate target gene.

The ticks were homogenized using a QIAGEN TissueLyzer (QIAGEN) and RNA extraction was performed in a QIAGEN M48 BioRobot using the MagAttract® RNA Tissue Mini M48 kit. Random hexamer primers and Illustra™ Ready-to-GO RT-PCR beads kit (GE Healthcare, UK) were used for cDNA synthesis. The cDNA was then used for analyzes of Bartonella spp. and tick identification.

A total of 386 tick specimens were analyzed for potential presence of Bartonella spp. RNA using a quantitative real-time PCR (q-PCR) targeting the citrate synthase gene (gltA) (7). The reaction was adjusted by using 5 µl cDNA template (instead of 1 µl). Negative and positive controls were included in each step, i.e. RNA extraction, cDNA synthesis, and q-PCR.

For the molecular identification of the 10 selected ticks, standard PCR amplifications were carried out in 25 µl reaction mixtures containing 5 µl of the cDNA, 1.65 mM MgCl2, 0.2 mM of the four dNTPs, 10 pM of each primer (T1B122S and T2A12S), 1 UTaq polymerase enzyme (Promega), and 1 µl Yellow SubTM (GENEO Bioproducts, Hamburg, Germany). The reaction mixture was overlaid by a drop of fine neutral mineral oil (ICN) and placed on a heating block of a programmable thermocycler (Biometra, Westburg). After a denaturation step of 4 min at 94°C, each of the 40 cycles consisted of 30 s at 92°C, 45 s at 58°C, and 60 s at 72°C before a final elongation step of 8 min at 72°C (8).

The PCR products were cloned prior to sequencing. For this, a TOPO TA Cloning® Kit was used (Invitrogen™). The clones thus obtained were sequenced by the VIB (Flemish Institute for Biotechnology) Genetic Service Facility at the University of Antwerp, using the ABI PRISM® BigDyeTM Terminator cycle sequencing kit and a capillary DNA sequencer (Applied Biosystems 3730 DNA Analyzer).

Results

One or more ticks were found on 2.7% of the birds, with means of 1.7 nymphs and 0.6 larvae, respectively, per infested bird (Table 1). The majority of the 386 ticks found were nymphs and larvae of Hyalomma (n=367; Table 2). Sequencing data for the 10 Hyalomma ticks revealed that nine were H. rufipes and one was H. marginatum. These findings supported the diagnoses based on tick morphology. In total, 119 (30.8%) ticks were larvae and 250 (68.1%) were nymphs, i.e. 98.9% of the ticks were larvae or nymphs. In the present study only one adult tick, a female I. ricinus, was found.

Table 1

Bird species infested with ticks during springtime migration

Scientific name	Common name	No. birds	No. ticks	No. (%) birds infested	Mean infestation rate (No. ticks/No. infested bird)	Mean no. larvae/infested bird	Mean no. nymphs/infested bird
Acrocephalus schoenobaenus	Sedge warbler	250	16	6 (2.4)	2.7	0.8	1.7
Acrocephalus scirpaceus	European reed warbler	9	4	1 (11)	4.0	0.0	4.0
Anthus trivialis	Tree pipit	208	11	7 (3.4)	1.6	0.1	1.3
Erithacus rubecula	European robin	52	5	2 (3.8)	2.5	0.0	2.5
Ficedula albicollis	Collared flycatcher	29	3	1 (3.4)	3.0	0.0	3.0
Ficedula hypoleuca	Pied flycatcher	1032	56	37 (3.6)	1.5	0.5	1.0
Hippolais icterina	Icterine warbler	292	4	4 (1.4)	1.0	0.8	0.3
Hippolais pallida	Eastern olivaceous warbler	24	1	1 (4.2)	1.0	0.0	1.0
Lanius senator	Woodchat shrike	53	28	7 (13)	4.0	0.9	3.1
Luscinia megarhynchos	Nightingale	118	15	6 (5.1)	2.5	2.3	0.2
Motacilla flava	Yellow wagtail	6	9	1 (17)	9.0	4.0	5.0
Muscicapa striata	Spotted flycatcher	572	2	2 (0.4)	1.0	0.5	0.5
Oenanthe oenanthe	Wheatear	4	2	2 (50)	1.0	0.0	1.0
Oriolus oriolus	Eurasian golden-oriole	148	12	7 (4.7)	1.7	0.3	1.4
Phoenicurus phoenicurus	Common redstart	176	20	12 (6.8)	1.7	0.2	1.5
Phylloscopus sibilatrix	Wood warbler	543	18	16 (2.9)	1.1	0.5	0.6
Phylloscopus trochilus	Willow warbler	464	3	3 (0.7)	1.0	0.3	0.7
Saxicola rubetra	Whinchat	745	80	38 (5.1)	2.2	0.9	1.2
Sylvia borin	Garden warbler	1005	6	4 (0.4)	1.5	1.0	0.3
Sylvia communis	Common whitethroat	863	85	42 (4.9)	2.1	0.4	1.6
Turdus philomelos	Song thrush	6	5	1 (17)	5.0	0.0	5.0
Other species		854	1*	0	0	0.0	0.0
	Total	7,453	386	200 (2.7)	1.9	0.6	1.7

*One tick was found on an unidentified bird species.

Table 2

Genus and stage of ticks

Tick genus	No. ticks	No. larvae (%)	No. nymphs (%)	No. adults	Unidentifiable
Hyalomma	369	117 (32%)	250 (68%)	–	2
Ixodes	7	1	5	1	–
Amblyomma	2	–	2	–	–
Haemaphysalis	2	–	2	–	–
Unidentifiable	6	1	4	–	1
Total	386	119 (30.8%)	263 (68.1%)	1	3

Bartonella spp. RNA was not detected in any of the 386 ticks analyzed.

Discussion

None of the 386 ticks collected from birds captured on Antikythera, Greece and Capri, Italy during April–May 2009 was positive for RNA of Bartonella. Adult ticks rarely infest small and medium-sized birds and, accordingly, 98.9% of the ticks in the present study were larvae and nymphs.

We could not find any published report on Bartonella infections in ticks collected from migratory birds. However, in agreement with the present results, Monks et al. (9) screened ticks collected from free-living and captive birds with suspected avian tick-related syndrome for the presence of zoonotic pathogens. All 161 ticks were negative for Bartonella DNA (9). Furthermore, 64 Carios capensis ticks from a brown pelican (Pelecanus occidentalis) rookery in South Carolina, US, were also negative for Bartonella DNA (10). Wild birds may be important hosts of several blood-feeding arthropods, including ticks, potentially infected with clinically important pathogens (4, 5). However, these published investigations on Bartonella and the present one do not support the notion of a geographic spread of Bartonella in ticks infesting migratory birds.

The proportion of ticks positive for Bartonella spp. DNA in other studies varies from very low, i.e. 0.43% in questing Amblyomma americanum in the southern United States (11) and 1.2% in I. ricinus ticks collected in the Czech Republic (12) to much higher in I. ricinus ticks collected from roe deer (Capreolus capreolus) in The Netherlands, where 60% of the ticks were positive for Bartonella spp. DNA (13). All 167 I. ricinus ticks collected by flagging vegetation in central Sweden were negative for Bartonella DNA (14). This could possibly be explained by the fact that 95% of the ticks were host-seeking larvae that had never taken a blood meal, and host-seeking nymphs that had previously (as larvae) taken one blood-meal.

In conclusion, the results of the present study provided no support to the hypothesis that ticks infesting springtime migratory birds may be infected with Bartonella bacteria. To our knowledge, this is the first published report about the potential presence of Bartonella bacteria in ticks carried by migratory birds.