We conducted fieldwork activities at the Ponza Ringing Station on the island of Ponza (Central Tyrrhenian Sea, Italy; 40°55¢N, 12°58¢E) during spring (March–May) 2016 and 2017. We captured 744 migratory birds belonging to 20 different species (Table) during regular ringing procedures and checked them for ticks. Fourteen bird species were long-distance migrants that wintered in sub-Saharan Africa, and 6 were partial migrants, such as the blackbird (Turdus merula), the dunnock (Prunella modularis), the Eurasian blackcap (Sylvia atricapilla), the European robin (Erithacus rubecula), the song thrush (Turdus philomenos), and the subalpine warbler (Sylvia cantillas).

We collected 231 engorged ticks and identified them using standard morphologic keys (3) and PCR amplification of the internal transcribed spacer (ITS) region when possible (4). We used commercial kits for RNA (High Pure Viral Nucleic Isolation Kit; Roche Diagnostics, https://diagnostics.roche.com) and DNA (High Pure PCR Template Preparation Kit; Roche Diagnostics) extraction. We used the RealStar CCHFV RT-PCR Kit 1.0 (Altona Diagnostics, https://www.altona-diagnostics.com) for CCHFV detection; we used conventional PCR with protocols described elsewhere (5) to detect DNA from Babesia spp., Anaplasma spp., Ehrlichia spp., SFG rickettsiae, and Borrelia spp. We used DNA from Babesia canis (dog 825/08, 1:10 diluted), Anaplasma phagocytophilum (cattle 2008/13, 1:10 diluted), Ehrlichia canis (clone), Rickettsia raoultii (clone) and B. burgdorferi (clone) as positive controls for each amplification.

Using PCR amplification of the ITS region, we identified 94 ticks at the species level: H. marginatum complex (5 larvae, 82 nymphs), I. frontalis (3 nymphs), I. ventalloi (3 nymphs), and Amblyomma marmoreum (1 nymph). Amplification of the ITS region failed in the remaining ticks, identifying only the genus; these ticks were mostly Hyalomma spp. (1 larva, 118 nymphs) or Ixodes spp. (3 larvae, 14 nymphs, and 1 adult).

Of the analyzed ticks, 50 tested positive for SFG rickettsiae DNA; the overall prevalence was 21.7% (95% CI 16.8%–27.4%). To determine the species, we amplified a fragment of the ompA gene in all the SFG rickettsiae–positive ticks (5). Positive amplicons were sequenced by LGC Genomics (https://www.lgcgroup.com) and compared with sequences deposited in GenBank. Results revealed R. aeschlimannii DNA in 47 (94.0% [95% CI 83.8%–97.9%]) of 50 ticks (Table). We identified 46 sequences identical to an R. aeschlimanni strain documented from Egypt (GenBank accession no. HQ335157), Turkey (GenBank accession no. MF379299), and Italy (GenBank accession no. MH532239) and 1 sequence identical to R. aeschlimanni strain RH (GenBank accession no. HM050286) from Senegal; the latter strain differed from the others by 1 nt (T instead of C at nt 425). Two (4.0% [95% CI 1.1%–13.5%]) sequences were identical to R. africae (GenBank accession no. HQ335132), and 1 (2.0% [95% CI 0.4%–10.5%]) sequence was identical to R. raoultii (GenBank accession no. MF166732). We also screened a subset of positive ticks using primers targeting a fragment of the gltA gene (5), confirming the results obtained with the ompA gene. No ticks tested positive for other microorganisms.

Although ticks of the H. marginatum species complex (i.e., H. marginatum, H. rufipes, H. turanicum, and H. isaaci) are the most widespread ticks in Africa, they also have been found in some countries in Europe, such as the United Kingdom (6). These tick species are also vectors for CCHFV, which occurs mainly in Africa and southeastern Europe and can cause life-threatening disease in humans. Hyalomma ticks are vectors and reservoirs of this virus; birds, which are the primary hosts for the immature stages of these ticks, can maintain and spread the virus into new areas through migration (7).

R. aeschlimannii and R. africae, which are zoonotic bacterial species endemic to Africa, are transmitted by ticks belonging to the genera Hyalomma and Amblyomma. However, these bacteria have also been detected in ticks from other regions, such as Oceania, the Caribbean islands, and Europe (7–9). Autochthonous cases of human rickettsiosis caused by R. aeschlimannii have been recently described in Greece (10) and Italy (11). We detected R. africae in H. rufipes and R. raoultii in Hyalomma sp., which are not known vectors for these pathogens. Because we did not test the birds for Rickettsia spp. and the ticks were engorged, we cannot exclude the possibility that the ticks acquired these microorganisms by feeding on positive birds. Nevertheless, our results agree with those observed in a study in Italy (12) and confirm the circulation of these Rickettsia species into areas to which they are not endemic. They also highlight role of migratory birds in the passive transportation of infected ticks.

Although no ticks tested positive for CCHFV in our study, some studies report this virus in H. marginatum complex ticks attached to birds migrating from Africa to Europe (13). Migratory birds might have contributed to the establishment of the CCHFV in Spain (14). Moreover, climate change could cause prolonged, warmer, and drier summers and autumns. These seasonal changes might lead to the establishment of autochthonous populations of Hyalomma ticks in areas previously free of these vectors. Finally, RNA of another relevant human pathogen, the recently discovered Alkhurma hemorrhagic virus (15), has been detected in ticks of the H. marginatum complex.

In summary, we found zoonotic bacteria in ticks carried by birds across their migratory routes and assessed the risk for pathogen introduction in Italy. However, further studies are needed to clarify the role of these ticks in the epidemiology of zoonotic pathogens.