Anaplasma phagocytophilum in Ticks Blood-Feeding on Migratory Birds in Sweden

Migratory birds play a dual role as potential reservoirs of tick-borne pathogens, and potential dispersers of pathogen-containing ticks during their migratory journeys. Ixodes ricinus, a prevalent tick species in Northern and Western Europe, serves as a primary vector for Anaplasma phagocytophilum—a bacterium with implications for human and animal health. There is limited information available regarding A. phagocytophilum in birds. Our investigation focused on A. phagocytophilum prevalence in ticks collected from migratory birds in southeastern Sweden. The identification of ticks involved both molecular analyses for species determination and morphological classification to ascertain the developmental stage. The presence of A. phagocytophilum was determined using real-time PCR. Of the 1115 ticks analyzed from 4601 birds, 0.9% (n = 10), including I. ricinus and Ixodes frontalis, tested positive for A. phagocytophilum. Notably, common blackbirds (Turdus merula) yielded the highest number of A. phagocytophilum-infected ticks. The findings suggest that A. phagocytophilum is present in a small proportion of ticks infesting migratory birds in southeastern Sweden. Consequently, the role of birds as hosts for ticks infected with A. phagocytophilum appears to be low, suggesting that birds seem to play a minor indirect role in the geographic dispersal of A. phagocytophilum.


Introduction
Birds are commonly infested with various tick species [1], and both birds and ticks may harbor potentially human-pathogenic bacteria, viruses, and protozoa [2,3].Since many birds are migratory, there is a potential risk for spread of pathogens to new areas along their migratory routes through ticks feeding on them [1,3].The prevalence of tick infestation varies considerably between different species of birds, where bird feeding behavior, geographical location, climate, and season are among the factors introducing variation [3].The highest prevalence of tick infestation is generally observed in groundforaging bird species, which is closely linked to tick exposure levels [4].Even though ticks can be transported by migratory birds, the establishment of new tick foci in an area depends on climate, vegetation, and the presence of suitable blood meal hosts, such as rodents [3].Previous studies of ticks on birds show that Ixodes ricinus is the most common tick species infesting birds in Europe [5] and that most ticks collected from birds belong to immature life stages, i.e., larvae and nymphs [2,5,6].However, it is important to note that several other tick species also feed on birds.Among them, ticks like Ixodes frontalis and Ixodes arboricola are ornithophagous [7].
Anaplasma phagocytophilum is an intracellular bacterium that has been detected in mammals, birds, and ticks [5].The bacterium uses mainly ticks in the I. ricinus complex as vectors, i.e., I. ricinus in Western Eurasia, Ixodes persulcatus in Eastern Eurasia, and Ixodes scapularis and Ixodes pacificus in North America [16].Domestic ruminants are commonly infected with A. phagocytophilum.In these hosts, the disease is called tick-borne fever (TBF) and is characterized by symptoms such as high fever, dullness, abortion, and a drop in milk production, which results in economic losses for livestock owners [17].Anaplasma phagocytophilum has also been detected in cats [18], dogs [19], birds [20], horses [21], and humans [22].In humans, the disease is called human granulocytic anaplasmosis (HGA), with symptoms ranging from nonspecific influenza-like symptoms such as fever, headache, myalgia, and malaise to severe illness [17].Cases of HGA have been described in Europe, North America, Asia, and Africa [16,17].The prevalence of A. phagocytophilum in fieldcollected I. ricinus varies among countries and locations with a prevalence of 12% in Sweden [23], 24% in Denmark [24], 14% in Poland [25], and 6% in Norway [26].The prevalence of A. phagocytophilum in I. ricinus ticks infesting birds is usually lower, below 5% [9,14,[27][28][29].Interestingly, the bacterium has also been detected in blood samples from passerine birds, suggesting that at least some birds might be capable of transmitting A. phagocytophilum to ticks [30].However, the importance of birds for the transmission cycle of A. phagocytophilum is currently unclear.
Investigating the prevalence of a particular tick-borne pathogen is essential, not only for addressing public health concerns and veterinary implications but also for advancing our understanding of ecological dynamics, recognizing seasonal variations, and providing geographically specific information about the microorganism within ticks.This study specifically aims to evaluate the prevalence of A. phagocytophilum in ticks infesting birds during their spring and autumn migrations in the southeastern region of Sweden.

Sampling, Analyses, and Processing of Ticks
For comprehensive details regarding the sampling site, bird capture procedures, bird classification, tick collection, and species determination, as well as nucleic acid extraction and cDNA synthesis, please refer to our previous report [8].
In summary, ticks were collected from birds captured between 15 March and 15 June, and between 15 July and 15 November 2009, at the Ottenby Bird Observatory, located on the southern tip of Öland Island in southeastern Sweden (56 • 12 N, 16 • 24 E).Each captured bird was identified to the species level.Subsequently, any ticks collected were photographed and morphologically classified according to their developmental stage (larva, nymph, or adult), and the sex of adults was determined.Individual ticks were homogenized using a TissueLyser II (Qiagen, Hilden, Germany), followed by extraction, purification, and isolation of total nucleic acids using the MagAttract ® Viral RNA M48 kit in a BioRobot M48 workstation (Qiagen, Hilden, Germany).The extracted nucleic acids were then reverse-transcribed to cDNA using the illustra™ Ready-to-Go RT-PCR Beads kit (GE Healthcare, Amersham Place, UK), serving as the template for all PCR assays.To determine the genus and species of the ticks, each specimen underwent PCR analysis targeting the tick mitochondrial 16S rRNA gene, followed by DNA sequencing.
The sampling of birds was approved by the Swedish Board of Agriculture, delegated through the Animal Research Ethics Committee in Linköping (decision 43-09).

Molecular Analyses of Anaplasma phagocytophilum in Ticks
Detection of A. phagocytophilum was carried out using a TaqMan real-time PCR assay, as previously described [31].The primers ApF (5 -TTT TGG GCG CTG AAT ACG AT-3 ) and ApR (5 -TCT CGA GGG AAT GAT CTA ATA ACG T-3 ) and the probe ApM (FAM-TGC CTG AAC AAG TTATG-BHQ1) are designed to target the A. phagocytophilum citrate synthase gene (gltA) to amplify a 64-bp long amplicon.A 25 µL reaction mixture comprised 12.5 µL Maxima ® Probe qPCR Master Mix (2×) (Thermo Fisher Scientific, Waltham, MA, USA), 1.5 µL of each primer and0.375 µL of probe (10 µM; Invitrogen, Waltham, MA, USA), 7.125 µL RNase-free water, and 2 µL cDNA template.PCR reactions were conducted using a C1000™ Thermal Cycler and CFX96™ Real-Time PCR Detection System from Bio-Rad Laboratories, Inc. (Hercules, CA, USA), with an initial activation step at 95 • C for 10 min, followed by 40 cycles of 95 • C for 15 s and 60 • C for 1 min.For quality control, a synthetic plasmid containing the target sequence of the TaqMan real-time PCR assay was employed as a positive control.This plasmid contained the target sequence, spanning nucleotides 304-420 of the A. phagocytophilum gltA gene (GenBank: AF304137), synthesized and cloned into the pUC57 vector (GenScript, Piscataway, NJ, USA).
Subsequent confirmation analysis was performed on samples positive in the TaqMan qPCR assay by conventional PCR, using the first primer pair of the A. phagocytophilum specific 16S PCR by Stuen et al. [32], producing an amplicon of 282 bp.Nucleotide sequencing of the PCR products was performed by Macrogen Inc. (Amsterdam, The Netherlands).All sequences obtained underwent confirmation by sequencing both strands.The resulting chromatograms were initially edited and analyzed using BioEdit Software v7.0 (Tom Hall, Ibis Therapeutics, Carlsbad, CA, USA), and the sequences were further scrutinized utilizing the Basic Local Alignment Tool (BLAST).

Ticks Collected from Birds
A total of 4601 bird individuals of 65 species were examined for ticks at least once during the study period at the Ottenby Bird Observatory.Among them, 749 bird individuals (comprising 759 bird captures) belonging to 35 species were found to be infested with a total of 1339 ticks.These findings were previously documented by Wilhelmsson et al. 2020, with detailed data available in Table 1 of the referenced publication [8].10) 1 The tick species and life stage remain undetermined, as the photograph of the tick is missing.Additionally, molecular identification was not carried out due to technical issues.
In this study, a total of 1115 ticks (cDNA samples) were available for analysis.Among these, 533 were larvae, 561 were nymphs, two were adult females, and 19 ticks could not be classified into a developmental stage due to technical constraints.The ticks subjected to analysis (Table 1) were identified through molecular methods, with 1042 identified as I. ricinus (consisting of 501 larvae, 534 nymphs, and seven specimens of unknown developmental stage), 23 as I. frontalis (comprising eight larvae, 14 nymphs, and one adult female), and 22 ticks designated solely as Ixodes spp.(including 12 larvae and ten nymphs).Additionally, 12 ticks were identified as Haemaphysalis punctata (eleven larvae, one nymph), and four ticks were recognized as Hyalomma marginatum (one larva, two nymphs, and one adult female).The remaining 12 ticks could not be molecularly identified to species or developmental stage due to inadequate sequences (despite multiple sequencing attempts) or the absence of accompanying photos.
The ticks analyzed in this study were removed from a total of 34 bird species (Table 2). 1 AP-positive = Anaplasma phagocytophilum-positive tick detected by real-time PCR.

Prevalence of Anaplasma phagocytophilum in the Ticks
Of the 1115 ticks analyzed, ten (0.9%) were detected positive for A. phagocytophilum by real-time PCR (Tables 1 and 2).Importantly, all infected ticks were removed from different individual birds.Six of the positive ticks were collected from common blackbirds (Turdus merula), two from European robins (Erithacus rubecula), one from a willow warbler (Phylloscopus trochilus), and one from a tree pipit (Anthus trivialis) (Tables 2 and 3).November I. frontalis N HF 1 NA, not available.The tick species, life stage, and the degree of blood engorgement remain undetermined, as the photograph of the tick is missing.Additionally, molecular identification was not carried out due to technical issues. 2The samples underwent prior analysis using real-time PCR to detect Borrelia burgdorferi sensu lato, as described in [8].Abbreviations: L, larva; N, nymph; LF, little fed; HF, half fed; FF, fully fed.
Seven of the A. phagocytophilum-positive ticks were I. ricinus (one larva and six nymphs), two were I. frontalis (one larva and one nymph), and one tick could neither be identified to species nor developmental stage (Table 3).
The ticks positive for A. phagocytophilum were mainly collected in April (n = 5) and most of them were half to fully fed (n = 7) (Table 3).According to the results of previous studies investigating the presence of Borrelia bacteria and Babesia protozoa in the same tick material as in the present study [8,12], three of the A. phagocytophilum-positive ticks were co-infected with Borrelia valaisiana (n = 1), Borrelia turdi (n = 1), and Borrelia miyamotoi (n = 1) (Table 3), but none was co-infected with Babesia spp.
We obtained DNA sequences from six out of the ten positive A. phagocytophilum samples.Four of these sequences were identical to each other and showed 100% sequence (257/257) identity with those of A. phagocytophilum (accession no.ON614171.1)detected in a tick collected in Serbia; one sequence showed 100% (260/260) sequence identity with A. phagocytophilum (accession no.MN252874.1)detected in a tick removed from a bird in Antikythira, Greece; and one sequence showed 99% (256/257) sequence identity with A. phagocytophilum (accession no.MT020436.1)that was found in various organs, including the heart, liver, spleen, lung, bone marrow, and blood-fed fleas and ticks collected from a Himalayan marmot (Marmota himalayana in China).Attempts to amplify the 16S rRNA from the remaining four real-time PCR-positive samples were unsuccessful.

Discussion
Our study revealed a 0.9% prevalence of A. phagocytophilum in ticks collected from migratory birds on the island of Öland in southeastern Sweden.Most infected ticks were nymphs of I. ricinus, predominantly found on common blackbirds, with the highest incidence occurring during the spring.Notably, three of the A. phagocytophilum-infected ticks were also co-infected with Borrelia bacteria.
Consistent with other European studies on ticks in birds, nearly all the ticks collected in our study were identified as I. ricinus [9,11,20,28].Moreover, all the I. ricinus ticks we encountered were subadults, aligning with previous research that highlights a preference among nymphs and larvae of I. ricinus for birds, particularly in ground-foraging species, as opposed to adult ticks that typically favor larger mammals and rarely feed on birds or small mammals [9,14,20,33,34].Only two adult female ticks were found, one I. frontalis and one H. marginatum.
Other tick species, like H. punctata and H. marginatum may also infest birds.However, these species were less common, and in total, less than 20 non-Ixodes ticks were found in our study.Adult H. punctata prefer medium to large mammals such as domestic ungulates and are rarely found on birds, while immatures are more commonly found on small mammals and birds [6,35,36].This was also the case in our study where most of the H. punctata were larvae.Every year, birds infested with H. marginatum are transported from Southern Europe to Sweden and other countries located on the same latitude [7].Birds and small mammals are known to host H. marginatum larvae and nymphs, while adult ticks prefer both wild and domestic ungulates [37].In our study, at least one tick of each developmental stage of H. marginatum was recorded, indicating that all stages can be transported to Sweden by birds.Previous studies have shown that H. punctata constitutes a minor risk in pathogen transmission from ticks to humans since this tick species rarely bite humans [7,36].Our results confirm other studies showing that H. marginatum ticks are not infected by A. phagocytophilum.This suggests that the interaction between H. marginatum ticks and migratory birds plays an insignificant role in the ecology of A. phagocytophilum [10,38].
Anaplasma phagocytophilum appears to be present in I. ricinus in all countries across Europe [17], with a prevalence range varying widely from 0.9% to 8.0% between different countries and studies [9,14,20,23,28].The prevalence among ticks collected from migratory birds in Southern Norway was 0.9% [14], consistent with our study's findings.In the present study, all ticks testing positive for A. phagocytophilum were removed from groundforaging birds, aligning with findings from previous studies highlighting the significance of ground-foraging birds, particularly thrushes, as key hosts for A. phagocytophilum-infected ticks [9,11,28].However, these findings could be attributed to the tendency of groundforaging bird species to contract a greater number of ticks, rather than indicating a preference of A. phagocytophilum for specific bird species.The presence of the bacterium has also been detected in blood samples from passerine birds, including Blackbird, Chaffinch, House Sparrow, Spanish Sparrow, Rock Bunting, Woodchat Shrike, Magpie, and Longtailed Tit [30].This suggests the possibility of certain avian species serving as hosts for transmitting A. phagocytophilum to ticks.Previous studies on the prevalence of A. phagocytophilum in ticks collected from migrating birds have demonstrated cases where larvae were infected with A. phagocytophilum [20,34], supporting the findings of our study where two Ixodes larvae were infected-one I. ricinus and one I. frontalis.Since transovarial transmission of A. phagocytophilum has not been observed [17,34], this implies that certain birds, particularly in terms of transmission to I. ricinus, might function as competent reservoirs or transmission hosts.Targeted investigations into avian blood could serve to delve deeper into this hypothesis.Nevertheless, the positive larvae in our study, as well as in other studies, could potentially have acquired their infections through tick co-feeding.
Three of the A. phagocytophilum infected ticks were co-infected by Borrelia species (B.valaisiana, B. miyamotoi, and Borrelia turdi, respectively).This is in line with previous studies on infected I. ricinus ticks collected from migrating birds in Switzerland and Norway presenting co-infection between B. garinii and A. phagocytophilum [9,14].Despite the large sample volumes, both in this study and in previous ones, the number of co-infected ticks remained low [9,14].However, co-infections must always be considered, especially in regions known for high prevalence of various tick-borne pathogens [39].Humans may be co-infected with tick-borne pathogens in two ways; either because of a tick bite from a multi-infected tick containing more than one pathogen, or because of more than one tick bite from ticks containing different pathogens [39].
While the presence of A. phagocytophilum in a tick is a concern due to its potential to transmit the bacterium to other hosts, it does not necessarily imply an immediate health risk for humans or other animals.The ability of A. phagocytophilum to infect different hosts and cause distinct diseases means that not all strains are equally virulent to humans or other species.The fact that A. phagocytophilum contains biologically distinct strains [40] that infect different host species, leading to human granulocytic anaplasmosis, equine granulocytic anaplasmosis, canine granulocytic anaplasmosis, and tick-borne fever, underscores the complexity of the interactions between the bacterium, its hosts, and the vectors involved.This diversity in strains and host specificity indicates that A. phagocytophilum has adapted to various mammalian hosts, and its pathogenicity may vary depending on the specific strain and host involved.Unfortunately, we did not conduct an analysis to characterize the strain of A. phagocytophilum detected in the present paper.
Another limitation of the study is the small number of Anaplasma-positive samples, coupled with the fact that the sampling took place 15 years ago, a constraint we acknowledge.Additionally, four out of ten A. phagocytophilum-positive specimens failed to amplify the 16S rRNA gene using conventional PCR assays.It is possible that these ticks contained a lower amount of A. phagocytopilum bacteria compared to ticks containing typeable A. phagocytophilum which may, at least partly, explain why PCR products, used to determine A. phagocytophilum, were not amplified in the conventional PCR assays.

Conclusions
This study reveals that the bacterium A. phagocytophilum is present in ticks infesting migratory birds both in spring and in autumn capture, in southeastern Sweden, which could lead to dissemination of these tick-borne microorganisms into new areas.However, the importance of birds as transmission hosts of A. phagocytophilum seems to be minor, although this is still unclear and needs further investigation.

Table 1 .
Presence of Anaplasma phagocytophilum in different tick species and life stages.

Table 2 .
Presence of Anaplasma phagocytophilum in ticks removed from different bird species.

Table 3 .
Characteristics of Anaplasma phagocytophilum-positive ticks removed from birds.