Tick Species Diversity and Molecular Identification of Spotted Fever Group Rickettsiae Collected from Migratory Birds Arriving from Africa

The role of migratory birds in the spread of ticks and tick-borne pathogens along their routes from Africa to Europe is increasingly emerging. Wild birds can host several tick species, often infected by bacteria responsible for zoonoses. The aim of the study is to assess the possible introduction of exotic ticks carried by migratory birds into Italy from Africa and to detect the presence of Rickettsia species and Coxiella burnetii they may harbor. During a two-year survey, we collected ticks from migratory birds captured during their short stop-over on Ventotene Island. Specimens were first identified by morphology or sequencing molecular targets when needed, and then tested by real-time PCR for the presence of selected pathogens. A total of 91% of the collection consisted of sub-Saharan ticks, more than 50% of which were infected by Rickettsia species belonging to the spotted fever group, mainly represented by R. aeschlimannii. In contrast, the suspected C. burnetii detected in two soft ticks were confirmed as Coxiella-like endosymbionts and not the pathogen. Although there are still gaps in the knowledge of this dispersal process, our findings confirm the role of migratory birds in the spread of ticks and tick-borne pathogens, suggesting the need for a continuous surveillance to monitor the potential emergence of new diseases in Europe.


Introduction
Ticks are blood-feeding ectoparasites and major vectors of pathogens that cause infectious diseases worldwide in humans and animals, including mammals, birds, and reptiles, representing one of the most important vectors of infectious zoonotic diseases. There are about 900 different species of ticks having a wide geographical distribution. These parasites can be passively transported for long distances through the movement of their hosts, contributing to the dissemination of tick-borne pathogens. Birds are among the vertebrate animal hosts with the greatest mobility capabilities that allow them to overcome geographical barriers and spread parasites at different scales [1]. In particular, migratory birds cover regional and intercontinental distances twice a year in a short time during their migratory seasons. Indeed, they may leave their European breeding grounds

Materials and Methods
The collection of ticks from birds was conducted during the spring seasons 2021 and 2022 within the framework of bird ringing (bird banding) activities on Ventotene island (coordinates 40 • 47 11 N, 13 • 24 55 E) located in the central Tyrrhenian sea (Figure 1). The island is one of the most important stop-over sites in the Mediterranean for migratory birds, mainly Passeriformes, and hosts a ringing station operating since 1988 as part of a large-scale and long-term project coordinated by the Italian Institute for Environmental Protection and Research (ISPRA) to monitor the spring migration. The sampling period started the end of March and continued until the end of May during the seasonal peak of trans-Saharan migratory birds' passage over the Island. It allowed us to focus on the collection of African ticks and TBPs transported from the endemic African areas. Birds were captured every day according to the weather conditions using nearly 350 m mist-net transects; they were handled for ringing procedures and checked for the presence of ticks. All the procedures were performed by authorized expert bird-ringers. Ticks were removed using a tick twister or tweezers and stored in Eppendorf tubes containing 70% ethanol at room temperature until processed. All the parasites collected from a single bird were stored in the same tube, recording the date and host species.

Materials and Methods
The collection of ticks from birds was conducted during the spring seasons 2021 and 2022 within the framework of bird ringing (bird banding) activities on Ventotene island (coordinates 40°47′11″ N, 13°24′55″ E) located in the central Tyrrhenian sea (Figure 1). The island is one of the most important stop-over sites in the Mediterranean for migratory birds, mainly Passeriformes, and hosts a ringing station operating since 1988 as part of a large-scale and long-term project coordinated by the Italian Institute for Environmental Protection and Research (ISPRA) to monitor the spring migration. The sampling period started the end of March and continued until the end of May during the seasonal peak of trans-Saharan migratory birds' passage over the Island. It allowed us to focus on the collection of African ticks and TBPs transported from the endemic African areas. Birds were captured every day according to the weather conditions using nearly 350 m mist-net transects; they were handled for ringing procedures and checked for the presence of ticks. All the procedures were performed by authorized expert bird-ringers. Ticks were removed using a tick twister or tweezers and stored in Eppendorf tubes containing 70% ethanol at room temperature until processed. All the parasites collected from a single bird were stored in the same tube, recording the date and host species.

Tick Identification and Pathogen Detection
Parasites were first classified according to life stage (larva, nymph, adult) and the morphologically was identified by using dichotomous published keys [23,24]. Ticks infesting migratory birds are mostly immature and their morphological identification at species level is often inconclusive. Therefore, they were individually subjected to nucleic acid extraction for molecular identification and screening for pathogen detection. Once taken out of the 70% ethanol, each tick was air-dried and homogenized in RLT lysis buffer provided by the BioSprint 96 One-For-All Vet kit (INDICAL BIOSCIENCE, Leipzig, Germany), using a 5 mm steel bead and Tissue-lyser LT (Qiagen, Hilden, Germany). The BS96 Vet 100 protocol for the extraction from animal tissues was applied according to the manufacturer's instructions.
The molecular identification of tick species was conducted through the amplification and sequencing of the following different molecular markers: 12S rRNA [25] for the identification of species belonging to the Hyalomma genus; 16S rRNA [26] for species belonging to the Ixodes genus; and a partial region of TROSPA gene [27] was sequenced to confirm the identification of Ixodes inopinatus specimens. Amblyomma and Argas specimens were characterized by analyses of 12S rRNA, 16S rRNA, cytochrome C oxidase subunit 1 (COI) gene [28], 18S rRNA, and 28S rRNA [29]. All PCR products were sent to Eurofins Genomics (https://eurofinsgenomics.eu/en/custom-dna-sequencing/ Last access: 13 June 2023) for sequencing. The obtained sequences were compiled using DS Gene v1.5 software (Accelrys Inc. 2003, San Diego, CA, USA) and analyzed using NCBI's Basic Local Alignment Search (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi, last access: 8 June 2023) for the identification of tick species. The presence of the pathogens' DNA was first tested with specific real-time PCR assays using published methods for Rickettsia sp. [30], R. aeschlimannii [31], and a screening assay for Coxiella burnetii [32], using the GoTaq real-time PCR Master Mix (Promega, Madison, WI, USA) and the instrument QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Samples positive for Rickettsia sp. were subsequently tested for R. aeschlimannii. Species identification of samples positive for Rickettsia sp. and negative for R. aeschlimannii was performed by the partial sequence analysis of OmpA and gltA in accordance with the protocol previously described [33]. PCR products were visualized by microfluidic electrophoresis TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA), purified using GeneAll Expin™ PCR SV columns, and sequenced by Eurofins Genomics (https://eurofinsgenomics.eu/en/custom-dna-sequencing/, last access: 3 July 2023). Sequences were then analyzed using SeqScape v3.0 software and compared with the GenBank database using the BLAST software (https://blast.ncbi.nlm.nih.gov/Blast.cgi, last access: 1 July 2023). The C. burnetii real-time PCR IS1111 [32] positive samples were then further investigated by 16S rDNA [34] partial sequence (1185 bp) analysis using MEGA11 version 11.0.11 [35], by the amplification of the ten specific markers [36] used for multi-spacers typing (MST) and through icd real-time PCR [37].

Tick Identification and Pathogen Detection
Most of the ticks were immatures, mainly represented by nymphs (67.1%) followed by larvae (32.5%), and only a few female adults (0.4%). The species identification of larvae and nymphs required the use of different molecular targets that assigned them to 11 different species (Table 2). Considering the geographical origin of the tick species identified, 90.9% (n = 1218/1340) were sub-Saharan collected from trans-Saharan migratory birds and two specimens of an intra-Palearctic migrant (common kestrel, Falco tinnunculus). Hyalomma rufipes represented the widely preponderant species in the collection (89.2% overall), followed by a small number of Amblyomma ticks, mainly belonging to the A. marmoreum complex and one A. variegatum. Six specimens were identified as H. truncatum, and three larvae were soft ticks (Argasidae) belonging to the Argas genus ( Table 2). One of these was identified as Ar. persicus (12S identity 100%, COI identity > 99%), while the other two, both collected from the same bird host in 2021, were described in a recent in-depth study [38] as belonging to a novel genotype strictly related to Ar. africolumbae. Finally, the remaining 9.1% of ticks (n = 122/1340) was represented by species commonly distributed in the Mediterranean Basin, found indiscriminately on both short-distance (No. ticks = 62) and long-distance migrants (No. ticks = 60) ( Table 2). Ticks positive in the real-time PCR screening for Rickettsia sp. were 53.8% (N = 721/1340). Among these, the great majority, 94.9% (N = 684/721), was identified as R. aeschlimannii and distributed among sub-Saharan ticks (96.20%; H. rufipes = 657/684, H. truncatum = 1/684), Mediterranean ticks (1.6%; H. marginatum = 11/684), and undefined species (2.2%; Hyalomma sp. = 14/684, Ixodes sp. =1/684). The DNA of the 37 specimens positive to Rickettsia sp. but negative to R. aeschlimannii was subjected to the amplification and sequencing of gltA and OmpA genes for species identification. Amplicons were obtained for 24 out of 37 positive ticks resulting in the identification of R. monacensis (n = 9), R. helvetica (n = 6), R. felis (n = 3), R. africae (n = 2), and two possible new species in Italy, namely, R. tamurae and R. asembonensis. However, for the latter species, the sequencing result also produced a high percentage identity with a rickettsial endosymbiont (Table 3). Only the two ticks genetically close to Ar. africolumbae assayed by real-time PCR targeting the IS1111 region for the presence of C. burnetii were positive (Ct 26.5-26.7). These results were also confirmed by the assay targeting the icd gene of the pathogen (Ct 31.9-32.4). On the other hand, C. burnetii MST markers were not amplified and the phylogenetic analysis of 16S partial sequence identified both samples as Coxiella-like endosymbionts ( Figure S1, Supplementary Materials).

Discussion
During the study, we collected ticks from migratory birds during their journey from Africa to Europe with the aim to detect the introduction of African tick species and related bacterial agents, focusing on novel Rickettsia species and C. burnetii. Most of the ticks belonged to African species and were collected from trans-Saharan migrants, clearly reflecting the original wintering areas of their avian hosts. The bacterial pathogen detection in ticks revealed a high rate of infection by several Rickettsia species, while two samples positive for C. burnetii in the IS1111 real-time PCR were subsequently identified as non-pathogenic Coxiella-like endosymbionts.
Interestingly, the most parasitized bird species, referring to the number of infested individuals and the number of ticks/bird, were the same seven species, all trans-Saharan. This result suggests a fundamental role of these few species in the transport and spread of ticks, probably due to their feeding behavior, to the abundance of ticks in the African wintering grounds or in their stop-over sites, which deserves an in-depth dedicated study. Most of the tick specimens collected from migratory birds on Ventotene during the project were from the genus Hyalomma and, particularly, belonged to the species H. rufipes, the most abundant and widespread tick species in sub-Saharan Africa, also present in small areas of North Africa [39]. Our results corroborate previous surveys on ticks infesting migratory birds [3][4][5][6][7][8] and also confirm the route of H. rufipes introduced in different countries in Central and Northern Europe in the last decade [9][10][11][12]40]. Furthermore, among the African ticks potentially at risk of introduction in Italy, the detection of specimens of the genus Amblyomma was of considerable interest since it was allochthonous for the whole European continent and a vector for several pathogens. The only species detected both on migratory birds and mammalian hosts in Italy was A. variegatum, which was first reported in Sicily in 1971, and later in Sardinia and Corsica in 2018 [41,42]. Similarly, another species, genetically close to A. marmoreum, has recently emerged from our study and from other different surveys on migratory birds [6,43]. The identification of these immature ticks assigned to the A. marmoreum complex is still debated as either A. marmoreum or A. nuttallii, due to the morphological and genetic inconsistencies of the adult-type specimens used as references [43]. The work to resolve the discrepancies is ongoing in cooperation with international experts. Both the species, widespread in central and southern Africa and typically associated with reptiles (mainly tortoises and monitor lizards) in the adult stage [44,45], have never been detected on Italian ground.
In addition, the finding of soft ticks of the genus Argas (Argasidae) on migratory species deserves our attention. These ticks, often found on poultry and synanthropic birds, are rarely found on actively migrating species because of their feeding behavior that implies multiple short meals, usually involving a change in host [46]. Nevertheless, during our collection, two trans-Saharan migratory birds were found parasitized by three larvae: one belonging to the species Ar. persicus, at present considered almost ubiquitous, and two genetically related to the African Ar. africolumbae. Our finding of Ar. persicus on birds during the stop-over on an Italian island enriched the exiguous data available for this species in Italy, described with few localized records in the last century [47] and recently described in the cavities/nests of trans-Saharan migratory birds in Tuscany [48]. Conversely, the two larvae, found during our collection and recently described in detail as a novel Argas genotype close to Ar. africolumbae by Menegon and colleagues [38], represent the first record of this tick in Italy. These results, together with the data from previous surveys [5], clearly indicate that intercontinental movements are a possible, though not frequent, route of the dissemination of soft tick species as well. On the other hand, migratory birds can be exposed to ticks also at stop-over sites during their journey, as demonstrated by the modest, but not insignificant, amount of Mediterranean tick species collected by trans-Saharan birds.
Among the TBPs present in Europe and Africa, rickettsiae are widespread in both the continents and are represented by an increasing number of species, whose ecology biology, epidemiology, geographical distribution, and potential pathogenicity are often still poorly understood [49]. As for the species commonly found on migratory birds, all the new tick species detected were found infected by pathogenic bacteria mainly belonging to the Rickettsia genus. Indeed, our results show a high prevalence (53.8%) of these bacteria in ticks transported by migrating avifauna, mostly represented by R. aeschlimannii, belonging to the SFGR and responsible for human diseases. This pathogen, mainly transmitted by ticks of the Hyalomma genus, is widespread in the African and Eurasian Continents, Italy included [17,50]. Despite the presence of R. aeschlimanni being documented in ticks collected from several Italian regions [51], the incidence of documented human cases is probably limited to cases with severe symptoms and underestimated due to the difficulties in providing a correct diagnosis. Moreover, the intercontinental movements of infected ticks through birds could potentially favor the introduction of new genotypes whose pathogenicity results may not yet be known. In addition to the species extensively described in Italy, such as R. monacensis, R. helvetica, and R. felis (Table 3), we found ticks infected by allochthonous Rickettsia species, some of which have recently been reported outside the traditional endemic areas of sub-Saharan Africa. These include R. africae, the etiological agent of African tick-bite fever, already reported in Italy in ticks carried by migratory birds [4,[6][7][8] and in the adults of A. variegatum found in Sardinia [42].
Of particular interest, however, is the detection of R. tamurae in a tick belonging to the A. marmoreum complex. This Rickettsia belongs to the human spotted fever group; however, pathogenicity in both animals and humans remains poorly understood. Its distribution is limited to the Asian continent and, to date, has been detected only in Japan and Korea [50]. R. tamurae was described for the first time in Japan in A. testudinarium in 1993, formally recognized as a novel species in 2006 [52], and then found often in association with the same tick species, typical of reptiles [53,54]. Thus, this represents the first detection of R. tamurae in Europe. In fact, although the detection of this species in Italy was reported in a recent systematic review [51], the authors of the cited study confirmed the identity of the detected bacteria, initially described as R. monacensis/R. tamurae, as R. monacensis [55]. It is not easy to explain how it could have come to Africa, and then to Europe via birds. However, we can assume a possible spread from Asia to Africa, not yet been documented, via the abundant migratory routes linking the two continents. The tree pipit (Anthus trivialis), the bird host of the R. tamurae-infected tick, might be a potential spreader having two main different "wintering" quarters, one in Africa and the other in India, reached by different populations spread over a broad breeding ground extending from Europe to all of central Asia [56]. Given the migratory flyways connection between the two continents traveled by hundreds of bird species [57], a possible first transmission of the pathogen in breeding areas, followed by the spread to other continents, cannot be ruled out. The limited studies on pathogen distribution in remote areas of Africa and central Asia explain this knowledge gap and emphasize the need for dedicated surveys. However, the common association of R. tamurae with ticks of the genus Amblyomma, parasites of reptiles in the adult stage, may suggest a kind of vector-pathogen specificity, further supporting our finding.
Conversely, the detection of a possible R. asembonensis in a soft tick (Argasidae) of the species Ar. persicus is yet to be confirmed. This pathogen has a wide distribution in the African continent and is typically transmitted by fleas; although, it has also been found in some ticks [58]. Nevertheless, its identification remains doubtful due to the high percentage of identity with a rickettsial endosymbiont and the lack of amplification and sequencing results from the OmpA gene.
Finally, we paid particular attention to the two Argas ticks that both produced positive results to C. burnetii by IS1111 real-time PCR. Since this target used for the detection of the pathogen is widespread in Coxiella-like endosymbionts of ticks [59], we investigated this by multiple approaches to shed light on the results obtained. The lack of amplification of C. burnetii MST markers and the phylogenetic relationship with Coxiella-like endosymbionts based on the 16S partial sequence analysis allowed us to assign the two samples to Coxiellalike and not C. burnetii. In this study, we also used icd to evaluate its potential use as a specific target for C. burnetii. Reeves and colleagues [60] reported the amplification of a 612-bp icd fragment in the Coxiella-like endosymbiont, while different results were achieved in a more recent study [37] where the icd target was not amplified from the panel of Coxiella-like infected ticks investigated. Based on our experience, a similar result can be ascribed to the low sensitivity of icd, a single copy target, rather than to the specificity of the test. In our study, indeed, we observed a mean shift of 5.6 cycles between the IS1111 and icd targets due to the different number of copies present in the genome. The icd is therefore unspecific as well as the IS1111 target, and thus useless for C. burnetii detection in ticks.

Conclusions
In this study, we provided a descriptive assessment of the potential arrival of allochthonous ticks and tick-borne bacterial agents, with a focus on Rickettsia species and C. burnettii, through migratory birds during their annual movements from Africa to Europe. The high percentage of immatures belonging to typical sub-Saharan tick species, mainly H. rufipes, confirmed the ability of long-distance migrants to introduce these arthropods into Europe. This occurrence, in light of the new environmental and ecological scenarios as consequences of global warming, which is rapidly changing the climate in the Mediterranean Basin, emphasized the increased risk of the introduction and spread of these vectors. In addition, the presence of as-yet undefined biological entities, such as those belonging to the A. marmoreum and Ar. africolumbae groups, and of other species not frequently encountered before, emphasized the knowledge gap concerning the diversity of African vectors, some of which are probably still unknown or not genetically characterized. Similarly, the high prevalence of bacterial pathogens in ticks, mainly belonging to the genus Rickettsia, pointed to a high level of circulation in the birds' wintering areas and consequently to a high risk of the spread of emerging species or genotypes whose pathogenicity results in humans and European fauna are still unknown. Conversely, pathogenic C. burnetii was not identified in any of the collected ticks.
In conclusion, although the key role of migratory birds in the dispersal of ticks and tick-borne pathogens between continents is evident at present, this natural process cannot be avoided. Moreover, the potential number of pathogenic parasites carried by the several tick species is very high; therefore, the research, through multi-target or metagenomics approaches, is encouraged to explore a wider range of TBD agents. Increasing surveillance in Italy and the Mediterranean Basin might be a key early warning method to promptly identify any tick-borne zoonotic diseases that pose threats to human and animal health.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/microorganisms11082036/s1, Figure S1: Phylogenetic tree of Coxiella sp. created from 16S partial sequence evolutionary analyses [35,61,62]. Funding: This research was in the framework of the project "Emerging and re-emerging zoonoses along the routes of migratory birds. An integrated approach to analyze the potential pathways of introduction and spread" (IZS AM 04/19 RC), funded by the Italian Ministry of Health.
Institutional Review Board Statement: Capture, handling, and sampling procedures were conducted by the staff of the Italian Institute for Environmental Protection and Research (ISPRA), under the authorization of Law 157/1992 (Art.4(1) and Art7(5)), which regulates the research on wild bird species. No approval from any ethical committee was needed.
Data Availability Statement: Data available upon request to the corresponding author.