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Lesiczkaetal. Parasites & Vectors (2023) 16:368
https://doi.org/10.1186/s13071-023-05996-7
RESEARCH
Circulation ofAnaplasma phagocytophilum
amonginvasive andnative carnivore species
living insympatry inPoland
Paulina Maria Lesiczka1*, Izabella Myśliwy2, Katarzyna Buńkowska‑Gawlik2, David Modrý1,3,4,
Kristýna Hrazdilová5,6, Joanna Hildebrand2 and Agnieszka Perec‑Matysiak2
Abstract
Background Anaplasma phagocytophilum is characterized by a worldwide distribution and distinguished from other
Anaplasmataceae by the broadest range of mammalian hosts and high genetic diversity. The role carnivores play
in the life cycle of A. phagocytophilum in Europe is uncertain. Currently, only the red fox is considered a suitable
reservoir host. In this study, we focused on native and invasive medium‑sized carnivore species that live in sympatry
and represent the most abundant species of wild carnivores in Poland.
Methods A total of 275 individual spleen samples from six carnivore species (Vulpes vulpes, Meles meles, Procyon lotor,
Nyctereutes procyonoides and Martes spp.) were screened combining nested PCR and sequencing for A. phagocyt-
ophilum targeting a partial groEL gene with subsequent phylogenetic analysis inferred by the maximum likelihood
method.
Results The DNA of A. phagocytophilum was detected in 16 of 275 individuals (5.8%). Eight unique genetic variants
of A. phagocytophilum were obtained. All detected haplotypes clustered in the clade representing European ecotype
I. Three variants belonged to the subclade with European human cases together with strains from dogs, foxes, cats,
and wild boars.
Conclusions While carnivores might have a restricted role in the dissemination of A. phagocytophilum due to their
relatively low to moderate infection rates, they hold significance as hosts for ticks. Consequently, they could contrib‑
ute to the transmission of tick‑borne infections to humans indirectly, primarily through tick infection. This underscores
the potential risk of urbanization for the A. phagocytophilum life cycle, further emphasizing the need for comprehen‑
sive understanding of its ecological dynamics.
Keywords Anaplasma phagocytophilum, Carnivores, Meles meles, Martes spp., Nyctereutes procyonides, Procyon lotor,
Vulpes vulpes, Invasive species
Open Access
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Parasites & Vectors
*Correspondence:
Paulina Maria Lesiczka
lesiczkapaulina@gmail.com
Full list of author information is available at the end of the article
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Lesiczkaetal. Parasites & Vectors (2023) 16:368
Background
Anaplasma phagocytophilum is a gram-negative alpha-
proteobacterium infecting neutrophils. It is character-
ized by a broad distribution [1, 2] and distinguished from
other Anaplasmataceae bacteria by the widest range of
mammalian hosts and high genetic diversity [3]. Based
on studies focused on ecology and genetic diversity, the
species of A. phagocytophilum consists of at least four
major ecotypes, of which only ecotype I has been proven
to infect humans in Europe so far [4, 5]. e main hosts
of ecotype I are ungulates [6, 7], dogs, cats, horses [8–
10], and various wild mammals in urban or suburban
areas, such as red foxes (Vulpes vulpes) [11–13], hedge-
hogs (Erinaceus sp.) [14–17], and wild boars (Sus scrofa)
[18–20]. European ecotype I of A. phagocytophilum is
mainly transmitted by the tick Ixodes ricinus, character-
ized by low host specificity [21, 22]. To some extent, the
nest-dwelling I. hexagonus, which is known to parasitize
hedgehogs, red foxes, and European badgers, is involved
in the circulation of ecotype I of A. phagocytophilum [23,
24]. Occasionally, species from other tick genera tested
positive for the presence of A. phagocytophilum DNA;
however, their significance is currently unknown [25–28].
In Europe, A. phagocytophilum has been detected by
molecular methods in wild carnivores from six fami-
lies: Canidae, Ursidae, Mustelidae (Caniformia), Felidae,
Procyonidae, and Viverridae (Feliformia) [3, 29–31].
Although the role of wild carnivores as reservoir hosts for
this pathogen in Europe is uncertain, some species such
as raccoon dogs and red foxes are capable of transmitting
A. phagocytophilum in nature. In this study, we focused
on native and invasive medium-sized carnivore species
living in sympatry and representing the most abundant
species of wild carnivores in Poland. us, the objec-
tives of this study were to understand the genetic diver-
sity of A. phagocytophilum in wild invasive and native
carnivores with overlapping ranges and to investigate
the possibility of cross-species transmission of genetic
variants (including zoonotic ones) of A. phagocytophilum
between these species.
Materials andmethods
Study area andsampling
e carcasses of red fox, raccoon dog, raccoon, badger,
and marten were collected in the forestry of Ruszów (51°
24′ 00.1″ N 15° 10′ 12.2″ E) in the Lower Silesia County
in Poland during the predator control, which was part of
the program for the reintroduction of capercaillie (Tetrao
urogallus) in the Lower Silesian Forest (project LIFE11
NAT /PL/428) in the years 2017–2019. All carcasses
were frozen and transported to the Department of Para-
sitology, University of Wrocław. A total of 275 individual
spleen samples from six carnivore species, red fox (V.
vulpes) (n = 48), raccoon dog (Nyctereutes procyonoides)
(n = 50), raccoon (Procyon lotor) (n = 42), badger (Meles
meles) (n = 51), beech marten (Martes foina) (n = 57), and
European pine marten (Martes martes) (n = 27) were col-
lected during necropsy. All samples were kept at − 20 °C
until further DNA isolation procedures.
DNA extraction, PCR protocols andsequencing
DNA was extracted from 10 mg of spleen using the
commercial GeneMatrix Bio-Trace DNA Purification
Kit (EURx, Poland) according to the manufacturer’s
instructions. PCRs for detection of A. phagocytophilum
were performed using 2 × PCRBIO Taq Mix Red (PCR
Biosystems, UK). To determine the groEL ecotype of
A. phagocytophilum, 1297 bp fragments of the groESL
operon or (in the case of a missing amplicon) 407bp of
the groEL gene were amplified by nested PCR as previ-
ously described [18]. To distinguish two marten species
(M. martes and M. foina) the rapid PCR–RFLP method
described by Vercillo etal. [32] was used.
Amplicons were separated by electrophoresis in a 1.5%
agarose gel stained with Midori Green Advance (Nip-
pon Genetics Europe, Germany) gel stain and visual-
ized under UV light. All PCR products of the expected
size were excised from the agarose gels, purified, and
sequenced in both directions using the amplification
primers. Sequencing was performed by Macrogen Capil-
lary Sequencing Services (Macrogen Europe, the Nether-
lands). e sequences obtained were processed using the
Geneious 11.1.4 software [33] and compared with those
available in the GenBank™ dataset by Basic Local Align-
ment Tool (BLAST).
Phylogenetic analysis
e phylogeny of A. phagocytophilum was constructed
using eight unique groEL haplotypes detected in this
study along with 65 sequences from GenBank, repre-
senting four ecotypes described by Jahfari etal. [4] and
a sequence from Anaplasma platys used as outgroup.
Due to unequal sequence lengths, the alignment was cal-
culated in two steps using the MAFFT algorithm ‘Auto’
strategy for sequences > 1000nt and the –add function
for implementing sequences < 1000nt in the alignment
with final length of 1402nt. e phylogenetic tree was
inferred by the maximum likelihood method by IQTREE
1.6.5 [34]. e best-fit evolution model was selected
based on the Bayesian information criterion (BIC) com-
puted by implemented ModelFinder [35]. Branch sup-
ports were assessed by the ultrafast bootstrap (UFBoot)
approximation [36] and by the SH-like approximate like-
lihood ratio test (SH-aLRT) [37]. Trees were visualized
and edited in FigTree v1.4.1 and Inkscape 0.91.
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Page 3 of 7
Lesiczkaetal. Parasites & Vectors (2023) 16:368
Results
e DNA of A. phagocytophilum was detected in 16 of
275 individuals (5.8%). e number of positive animals
per species ranged from one (2%) in raccoon dog to five
(8.8%) in beech marten (Table1). ree long (> 1000nt)
and 13 short (300–400nt) sequences of the groEL gene
representing 8 unique genetic variants were obtained.
e major genetic variant V1 was detected in seven sam-
ples derived from four martens and a single European
badger, red fox, and raccoon, respectively. Two other
variants, V2 and V3, were detected in two animals each.
Variant V2 was found in red fox and racoon dog, and var-
iant V3 was detected in samples from red foxes only. e
remaining variants V4–V8 were detected in one sample
each from three martens, one badger, and one raccoon
(Table1). e representative sequences were submitted
to the GenBank under the accession number OR167090-
OR167101. In phylogenetic analyses (Fig.1), all detected
haplotypes clustered in the largest clade representing
European ecotype I [4], which is closely related to iso-
lates from the USA and forms cluster I [5]. ree variants
(V1, V3, and V8) belonged to the subclade with Euro-
pean human cases and strains from dogs, foxes, cats, and
wild boars. e remaining five variants were distributed
among strains isolated from I. ricinus, European hares,
carnivores, and sequences obtained from ungulates.
Discussion
e persistence and transmission of tick-borne patho-
gens in ecosystems relies upon abundance of susceptible
reservoir hosts and their infestation by permissive tick
species. Studies on European strains of A. phagocytophi-
lum have shown that a wide range of animal species are
involved in the circulation of this pathogen in different
ecological niches [38]. Among all Anaplasma spp., A.
phagocytophilum represents an assemblage with enor-
mous genetic diversity. Clarifying which host species
harbor specific strains of Anaplasma is important for
understanding pathogen dynamics and for developing
measures to reduce disease burden [39]. e role of car-
nivores in the ecoepidemiology of A. phagocytophilum is
not well understood. While several wild carnivores have
been implicated as possible reservoirs for A. phagocyt-
ophilum in the US, only the red fox has been considered
a suitable host in Europe [12, 29, 30, 40–42]. Carnivores
such as badgers and martens are often overlooked in
studies. is information gap also affects invasive species
such as raccoons and raccoon dogs, which were inten-
tionally introduced to Europe and later spread through
the continent [43]. In our study, we have shown that both
native (foxes, badgers, martens) and invasive (raccoons)
carnivores living in sympatry in a forest biotope are
involved in the circulation of A. phagocytophilum with
zoonotic potential, finding the genetic variant V1 in all
examined species except raccoon dogs (Table1, Fig.1).
e red fox is the most widespread free-living preda-
tor in the world [44], and its role as a host for A. phago-
cytophilum is well documented [45, 46]. In Poland, A.
phagocytophilum has been detected in foxes with preva-
lence ranging from 2.7% in the central part of the coun-
try [11] to 34.5% in the northeastern regions [31]. In our
study, 6.2% of animals tested positive for this pathogen,
which is consistent with the general trend observed for
Anaplasma infections in the European fox population
and supports foxes as a reservoir of A. phagocytophilum.
Only a few previous studies have focused on the role
of mustelids in the circulation of A. phagocytophilum. In
this study, 3.9% of badgers and 9.5% of martens were pos-
itive for A. phagocytophilum DNA. Analyses focused on
badgers and martens from eastern and northern Poland
detected the DNA of A. phagocytophilum in 18.7% and
41.7% of animals, respectively [31]. For comparison, the
number of positive badgers from Spain and e Nether-
lands did not exceed 2% [39, 47]. Data on A. phagocyt-
ophilum in European marten populations are sparse. To
our knowledge, this pathogen has been detected so far
in a beech marten from Romania [28] and a pine mar-
ten from Hungary [45] in which ecotype I was recog-
nized [4]. In addition, in mustelids from e Netherlands
tested by quantitative polymerase chain reaction (qPCR)
for several Tick Borne Pathogens (TBPs), A. phagocyt-
ophilum was detected in beech martens (1.5%), European
Table 1 The prevalence of Anaplasma phagocytophilum among invasive and native carnivore species living in sympatry in Poland
a All genetic variants detected in this study belong to ecotype-I [4]
Species Total number of animals No. of positive animals/prevalence Genetic varianta
Red fox (Vulpes vulpes) 48 3/6.2% V1, V2, V3
Raccoon (Procyon lotor) 42 2/4.7% V1, V8
Raccoon dog (Nyctereutes procyonoides) 50 1/2% V2
Beech marten (Martes foina) 57 5/8.8% V1, V6
European pine marten (Martes martes) 27 3/11% V1, V5, V7
Meles meles (Meles meles) 51 2/3.9% V1, V4
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Page 4 of 7
Lesiczkaetal. Parasites & Vectors (2023) 16:368
badgers (1.8%), European polecats (Mustela putorius)
(4.9%), and pine martens (22%) [39]. e observed dis-
crepancies in overall prevalence are likely due to the spe-
cific environmental conditions under which each study
was conducted, affecting tick occurrence and density.
e type of tissue and molecular method used to detect
pathogens may also explain the differences in results [17].
e results of our study indicate that martens are signifi-
cantly more susceptible to Anaplasma infection, with a
consistent increase in prevalence observed in these pred-
ators in all cited studies. e differences in distribution
patterns between the two species (the pine marten has a
patchy, fragmented ecogeographic distribution restricted
to a narrow ecological niche, whereas the beech mar-
ten has a continuous distribution across a wide range of
natural, semi-natural, and even urban habitats) may have
implications for the ecoepidemiology of A. phagocytophi-
lum, particularly in the context of rapid landscape change
0.06
0.04
MK069936 Ixodes ricinus Norway
GQ452228 Bos taurus Switzerland
MG670108 Procyon lotor Poland
V8
AF172163 Homo sapiens USA
MT498616 Sus scrofa Czech Republic
KU712090 Canis familiaris Hungary
LC167304 Homo sapiens Netherlands
V5
V6
ON153215 Vulpes vulpes Czech Republic
JF494839 Homo sapiens USA
MG570466 Homo sapiens Poland
AF033101 Homo sapiens Slovenia
KU712132 Vulpes vulpes Germany
AF172159 Homo sapiens USA
KU712086 Felis catus Finland
KJ832487 cattle France
MW762533 Lepus europaeus Czech Republic
V3
V2
V4
KF015601 Homo sapiens Poland
V7
V1
83.9/79
77.7/86
96/97
M. meles
V. vulpes, Martes sp., M. meles, P. lotor,
V. vulpes, N. procyonoides V. vulpes,
P. lotor
V. vulpes
Martes foina
Martes martes
Martes martes
Ecotype I
USA variant
Ecotype I
Ecotype II
Ecotype III
Ecotype IV
I. ventalloi
A
C
B
97.1/100
99.8/100
73.7/82
75/71
87.7/69
97.4/85
95.3/89
98.8/100
76.3/75
88.4/97
55.4/74
100/100
Fig. 1 A Schematic representation of the maximum likelihood phylogenetic tree based on the groEL gene sequences of Anaplasma
phagocytophilum representing all ecotypes. The highlighted clade representing Ecotype I is displayed in detail; bootstrap values (SH‑aLRT/
UFB) above the 70/70 threshold are displayed; sequence of Anaplasma platys used as an outgroup is not shown. B Detailed view of the clade
representing the Ecotype I/Cluster I; sequences acquired from the GenBank database are marked by their accession number, host, and country
of origin. Sequences from this study are highlighted in red and marked by the number of a respective variant. The scale bar indicates the number
of nucleotide substitutions per site. C Map of Poland with a detailed locality of Ruszów Forestry sampling area
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 5 of 7
Lesiczkaetal. Parasites & Vectors (2023) 16:368
and intense urbanization processes. Nevertheless, the
current lack of comprehensive studies makes it difficult
to fully elucidate the relationships. Determining whether
martens have exclusive host/reservoir competence for
Anaplasma, Anaplasmataceae, or other tick-borne path-
ogens is a complex task that requires further investiga-
tion. Invasive carnivore species, such as raccoons and
raccoon dogs, are potential reservoirs for numerous
TBPs [43, 48], and we also found A. phagocytophilum in
2% of raccoon dogs and 4.7% of raccoon dogs. e preva-
lence of A. phagocytophilum previously observed in rac-
coon dogs from Poland (35.3%) [31] was higher than in
Germany (23%) [47]. Kjær and colleagues reported a high
clustering of A. phagocytophilum-positive ticks on indi-
vidual raccoon dogs in Denmark [49]. Raccoons from
Austria, the Czech Republic, Germany, and Poland [50,
51] were tested for the presence of A. phagocytophilum
DNA, but the pathogen was found in only one raccoon
from the latest study [43]. Our results show that raccoons
are adapted to carry European variants of A. phagocyt-
ophilum. Due to their synanthropic nature and frequent
use of tree holes and burrows of other animal species,
raccoons can be infested with both questing and endo-
philic ticks, potentially bridging the enzootic cycles of A.
phagocytophilum. Regarding the epidemiological impact
of raccoons and raccoon dogs, these invasive species
should be monitored for their possible involvement in
the spread of A. phagocytophilum in different geographic
regions [51].
In recent years, awareness of role of wildlife in TBPs
and the possible impact on livestock, humans, and their
pets has increased [52]. Knowledge of potential reser-
voir hosts and their ticks is necessary to develop effec-
tive surveillance and management measures for disease
outbreaks and parasite cycles in wildlife [53]. Nidicol-
ous ticks such as Ixodes hexagonus, which are commonly
found on foxes and have been detected on mustelids [39,
54], deserve future attention as they may play a role as
vectors for zoonotic variants of A. phagocytophilum. In
addition, high population densities of predator popula-
tions are possible in European landscapes with hetero-
geneous habitat structure, leading to shared territories
among red foxes, raccoons, and raccoon dogs [55–57],
favoring the transmission of vectors and pathogens. e
increasing distribution and numbers of foxes in urban
and suburban areas make this species a bridging species
between natural ecosystems and anthropogenic land-
scapes [39].
Conclusions
While carnivores might have a restricted role in the dis-
semination of A. phagocytophilum due to their relatively
low to moderate infection rates, they hold significance
as hosts for ticks. Consequently, they could contribute to
the transmission of tick-borne infections to humans indi-
rectly, primarily through tick infection. is underscores
the potential risk of urbanization for the A. phagocytophi-
lum life cycle, further emphasizing the need for compre-
hensive understanding and management of its ecological
dynamics.
Acknowledgements
The carnivores’ carcasses were collected during the predator control operation
conducted as a part of the program to re‑introduce the capercaillie (Tetrao
urogallus) in the Lower Silesian Forest financed by the European Commission,
the National Fund for Environmental Protection and Water Management, and
the Polish State Forests (Grant LIFE11 NAT/PL/428). We are grateful to Janusz
Kobielski and Marcin Popiołek, PhD, DSc, for their help in collecting the mate‑
rial. We thank Weronika Hildebrand, DVM, for help with laboratory work.
Author contributions
PL, KH: methodology; PL, IM: formal analysis; KH, JH, APM: funding acquisi‑
tion: PL, KH: original draft writing; all authors were responsible for editing and
review; all authors approved the version to be submitted.
Funding
KH was supported by the project National Institute of Virology and Bacteriol‑
ogy (Programme EXCELES, ID Project No. LX22NPO5103)—Funded by the
European Union—Next Generation EU.
Data availability
Data will be made available on request.
Declarations
Ethics approval and consent to participate
The approval of the Ethics Committee was not required because the material
for the research was obtained from the predator control operation.
Competing interests
The authors declare no conflict of interest.
Author details
1 Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural
Resources, Czech University of Life Sciences Prague, Prague, Czech Repub‑
lic. 2 Department of Parasitology, Faculty of Biological Sciences, University
of Wrocław, Wrocław, Poland. 3 Biology Centre, Institute of Parasitology, Czech
Academy of Sciences, České Budějovice, Czech Republic. 4 Department
of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech
Republic. 5 Faculty of Medicine in Pilsen, Biomedical Center, Pilsen, Czech
Republic. 6 Department of Chemistry and Biochemistry, Mendel University,
Brno, Czech Republic.
Received: 26 June 2023 Accepted: 4 October 2023
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