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Circulation of Anaplasma phagocytophilum among invasive and native carnivore species living in sympatry in Poland

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Parasites & Vectors
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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. phagocytophilum 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 contribute 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 comprehensive understanding of its ecological dynamics. Graphical Abstract
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Lesiczkaetal. Parasites & Vectors (2023) 16:368
https://doi.org/10.1186/s13071-023-05996-7
RESEARCH
Circulation ofAnaplasma phagocytophilum
amonginvasive andnative carnivore species
living insympatry inPoland
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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Page 2 of 7
Lesiczkaetal. 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) [1113], hedge-
hogs (Erinaceus sp.) [1417], and wild boars (Sus scrofa)
[1820]. 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 [2528].
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, 2931].
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 andmethods
Study area andsampling
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 andsequencing
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) 407bp 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 etal. [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 etal. [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 > 1000nt and the –add function
for implementing sequences < 1000nt in the alignment
with final length of 1402nt. 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
Lesiczkaetal. 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 (Table1). ree long (> 1000nt)
and 13 short (300–400nt) 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
(Table1). 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, 4042]. 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 (Table1, 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
Lesiczkaetal. 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
Lesiczkaetal. 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 [5557],
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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... The bacterium Anaplasma phagocytophilum is transmitted by ticks and can infect various mammals, including humans 13 . In raccoons, it has been detected with varying prevalence [14][15][16][17][18] . Other bloodassociated bacteria detected in raccoons include Ehrlichia spp., Bartonella spp., and Rickettsia spp. ...
Article
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Raccoons (Procyon lotor) originated in North America and have been introduced to Europe. Due to their close contact with human settlements, they are important reservoirs for zoonotic pathogens, such as Baylisascaris procyonis. The relevance and prevalence of vector-borne pathogens have not yet been fully elucidated. In this study, we screened 285 spleen samples of raccoons, collected between 2019 and 2022 in Germany. The samples were analysed by PCR to detect Mycoplasma spp., Anaplasmataceae, Bartonella spp., Babesia spp., Rickettsia spp., Filarioidea, Trypanosomatida and Hepatozoon spp., and positive PCR products were sequenced. In total, 104 samples were positive for Mycoplasma spp. (36.49%), making this the first study to detect Mycoplasma spp. in raccoons outside of North America. Three samples were positive for Babesia spp. (1.05%) and two for Anaplasma phagocytophilum (0.7%). Phylogenetic analysis revealed that the Mycoplasma spp. detected all belong to the haemotrophic mycoplasmas cluster and were grouped within a single phylogenetic clade. Two different Babesia spp. were detected, one of which was closely related to Babesia canis, while the other was more closely related to Babesia sp. from ruminants. It is unclear whether the pathogens detected have an impact on the health of raccoons or whether they may serve as a reservoir for other animals.
... In dogs, B. vulpes and badger-associated Babesia sp. can cause severe disease including anaemia and thrombocytopenia (Miró Table 1 Detection of tick-borne pathogen nucleic acid in ticks collected from red foxes in Great Britain (n = 93) Hornok et al., 2018;Unterköfler et al., 2023), and A. phagocytophilum can cause polyarthritis and thrombocytopenia (Martinescu et al., 2023). Although B. vulpes and badger-associated Babesia are considered non-zoonotic (Azagi et al., 2021) an A. phagocytophilum variant (ecotype 1) with zoonotic potential to cause human granulocytic anaplasmosis has been detected in European foxes (Lesiczka et al., 2023b). ...
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Background Ixodes inopinatus was described from Spain on the basis of morphology and partial sequencing of 16S ribosomal DNA. However, several studies suggested that morphological differences between I. inopinatus and Ixodes ricinus are minimal and that 16S rDNA lacks the power to distinguish the two species. Furthermore, nuclear and mitochondrial markers indicated evidence of hybridization between I. inopinatus and I. ricinus. In this study, we tested our hypothesis on tick dispersal from North Africa to Southern Europe and determined the prevalence of selected tick-borne pathogens (TBPs) in I. inopinatus, I. ricinus, and their hybrids. Methods Ticks were collected in Italy and Algeria by flagging, identified by sequencing of partial TROSPA and COI genes, and screened for Borrelia burgdorferi s.l., B. miyamotoi, Rickettsia spp., and Anaplasma phagocytophilum by polymerase chain reaction and sequencing of specific markers. Results Out of the 380 ticks, in Italy, 92 were I. ricinus, 3 were I. inopinatus, and 136 were hybrids of the two species. All 149 ticks from Algeria were I. inopinatus. Overall, 60% of ticks were positive for at least one TBP. Borrelia burgdorferi s.l. was detected in 19.5% of ticks, and it was significantly more prevalent in Ixodes ticks from Algeria than in ticks from Italy. Prevalence of Rickettsia spotted fever group (SFG) was 51.1%, with significantly greater prevalence in ticks from Algeria than in ticks from Italy. Borrelia miyamotoi and A. phagocytophilum were detected in low prevalence (0.9% and 5.2%, respectively) and only in ticks from Italy. Conclusions This study indicates that I. inopinatus is a dominant species in Algeria, while I. ricinus and hybrids were common in Italy. The higher prevalence of B. burgdorferi s.l. and Rickettsia SFG in I. inopinatus compared with that in I. ricinus might be due to geographical and ecological differences between these two tick species. The role of I. inopinatus in the epidemiology of TBPs needs further investigation in the Mediterranean Basin. Graphical Abstract
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Background The raccoon Procyon lotor (Linnaeus, 1758) (Carnivora; Procyonidae) is one of the most important and most intensively studied invasive mammal species in Europe. Within the last 30 years the raccoon has spread at an increasing rate, resulting in the establishment of local populations in various regions of Europe. In these newly colonised areas, gaps in knowledge of the raccoon’s biology concern not only most aspects of its ecology in a broad sense, but also its pathogens and parasites. Most micropathogens recorded hitherto in the raccoons that have colonised Europe have documented epizootic and zoonotic potential. Thus, it is considered especially important to investigate the role played by the raccoon in the spread of pathogens through both animal-animal and animal-human pathways. Methods Tissue samples of raccoons from Poland and Germany were examined in this study. In total, 384 tissue samples from 220 raccoons (170 spleen samples, 82 liver biopsies, 132 ear biopsies) were examined using molecular methods. The presence of Rickettsia spp. DNA was screened through amplification of a fragment of the gltA gene. Samples that were PCR positive for gltA were tested for other rickettsial genes, ompB and a 17-kDa antigen. For taxonomic purposes, the obtained sequences were compared with corresponding sequences deposited in GenBank using the Basic Local Alignment Search Tool, and phylogenetic analyses were conducted using Bayesian inference implemented in MrBayes software. Results Rickettsia DNA was confirmed only in skin biopsies; no isolates from the spleen or liver were positive for Rickettsia DNA. With the exception of one sample from Germany, which was positive for Rickettsia helvetica DNA, all the samples positive for Rickettsia DNA derived from the Polish population of raccoons. DNA of Rickettsia spp. was detected in 25 samples, i.e. 11.4% of the tested raccoons, and R. helvetica was confirmed in 52% of the positive samples. Additionally, single cases of Rickettsia monacensis , Rickettsia raoultii , and Candidatus Rickettsia kotlanii-like were found, and in 32% of all the positive samples similarity was shown to different Rickettsia endosymbionts. Out of the samples that tested positive for gltA , amplicons of ompB and 17 kDa were successfully sequenced from 14 and three samples, respectively. Conclusions To the best of our knowledge, this study provides, for the first time, evidence of the occurrence of Rickettsia pathogens and endosymbionts in the European population of raccoons. Further, broader research on different species of wild vertebrates, and ticks, as potential vectors and hosts for tick-borne pathogens, in natural as well as in peri-urban environments, is therefore required. Graphical abstract
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In recent decades, populations of the raccoon ( Procyon lotor ) and the raccoon dog ( Nyctereutes procyonides ) have increased and adapted to peri-urban and urban environments in many parts of the world. Their ability to rapidly colonize new territories, high plasticity and behavioral adaptation has enabled these two species to be considered two of the most successful invasive alien species. One of the major threats arising from continually growing and expanding populations is their relevant role in maintaining and transmitting various vector-borne pathogens among wildlife, domestic animals and humans. According to the WHO, over 17% of infectious diseases are vector-borne diseases, including those transmitted by ticks. Every year tick-borne pathogens (TBPs) create new public health challenges. Some of the emerging diseases, such as Lyme borreliosis, anaplasmosis, ehrlichiosis, babesiosis and rickettsiosis, have been described in recent years as posing important threats to global health. In this review we summarize current molecular and serological data on the occurrence, diversity and prevalence of some of the TBPs, namely Babesia , Theileria , Hepatozoon , Borrelia , Rickettsia , Bartonella , Anaplasma and Ehrlichia , that have been detected in raccoons and raccoon dogs that inhabit their native habitats and introduced areas. We draw attention to the limited data currently available on these invasive carnivores as potential reservoirs of TBPs in different parts of the world. Simultaneously we indicate the need for more research in order to better understand the epidemiology of these TBPs and to assess the future risk originating from wildlife. Graphical Abstract
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Hedgehogs are small insectivorous mammals common across Europe, Asia, and Africa. The increased encroachment of humans into hedgehog habitats has disrupted the human-animal-environment interface. With growing interest in the zoonotic diseases of wildlife species, more studies have been devoted to this subject in the last few years. These papers provide information about known and new emerging diseases. Here we review the current knowledge regarding bacterial, viral, protozoic, and mycotic pathogens with zoonotic potential and assess the importance of hedgehogs as their carriers. Both wild and pet hedgehogs were included in the review. Data from several countries and various hedgehog species were included. The study shows the importance of hedgehogs as carriers of zoonotic diseases and reservoirs of zoonotic pathogens in varied habitats.
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Anaplasma phagocytophilum is an important tick-borne zoonotic agent of human granulocytic anaplasmosis (HGA). In Europe, the Ixodes ticks are the main vector responsible for A. phagocytophilum transmission. A wide range of wild animals is involved in the circulation of this pathogen in the environment. Changes in populations of vertebrates living in different ecosystems impact the ecology of ticks and the epidemiology of tick-borne diseases. In this study, we investigated four species, Western European hedgehog (Erinaceus europaeus), northern white-breasted hedgehog (Erinaceus roumanicus), Eurasian red squirrel (Sciurus vulgaris), and the common blackbird (Turdus merula), to describe their role in the circulation of A. phagocytophilum in urban and periurban ecosystems. Ten different tissues were collected from cadavers of the four species, and blood and ear/skin samples from live blackbirds and hedgehogs. Using qPCR, we detected a high rate of A. phagocytophilum: Western European hedgehogs (96.4%), northern white-breasted hedgehogs (92.9%), Eurasian red squirrels (60%), and common blackbirds (33.8%). In the groEL gene, we found nine genotypes belonging to three ecotypes; seven of the genotypes are associated with HGA symptoms. Our findings underline the role of peridomestic animals in the ecology of A. phagocytophilum and indicate that cadavers are an important source of material for monitoring zoonotic pathogens. Concerning the high prevalence rate, all investigated species play an important role in the circulation of A. phagocytophilum in municipal areas; however, hedgehogs present the greatest anaplasmosis risk for humans. Common blackbirds and squirrels carry different A. phagocytophilum variants some of which are responsible for HGA.
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The red fox (Vulpes vulpes) is the most widespread free-living carnivore in the world. Over the years, foxes have been recognized as hosts for a number of tick-borne pathogens. However, their role as reservoirs for zoonotic tick-borne diseases is poorly understood. The aim of our study was to investigate tick-borne pathogens in the red fox population in the Czech Republic. Out of 117 red foxes, 110 (94.02%) individuals tested positive for the presence of at least one pathogen by the combined PCR and sequencing approach. Hepatozoon canis was the most frequently detected pathogen (n=95; 81.2%), followed by Babesia vulpes (n=75; 64.1%). Babesia canis was not detected in our study. Four (3.42%) red foxes were positive for Candidatus Neoehrlichia sp., 3 (2.56%) for Anaplasma phagocytophilum, and one red fox (0.85%) tested positive for the presence of Ehrlichia sp. DNA. Overall, DNA of spirochetes from the Borrelia burgdorferi s.l. complex was detected in 8.6% of the foxes and B. miyamotoi in 5.12% of the samples. As a carnivore found in all ecosystems of Central Europe, foxes obviously contribute to transmission of tick-borne pathogens such as A. phagocytophilum, B. burgdorferi s.l., and B. myiamotoi. In addition, foxes apparently harbour a community of pathogens, associated with this host in local ecological context, dominated by H. canis and B. vulpes (possibly also Candidatus Neoehrlichia sp.). These species have the potential to spread to the domestic dog population and should be included in the differential diagnosis of febrile diseases with hematologic abnormalities in dogs.
Article
The genus Anaplasma (family Anaplasmataceae, order Rickettsiales) includes obligate intracellular alphaproteobacteria that multiply within membrane-bound vacuoles and are transmitted by Ixodidae ticks to vertebrate hosts. Since the last reclassification of Anaplasmataceae twenty years ago, two new Anaplasma species have been identified. To date, the genus includes eight Anaplasma species (A. phagocytophilum, A. marginale, A. centrale, A. ovis, A. bovis, A. platys, A. odocoilei, and A. capra) and a large number of unclassified genovariants that cannot be assigned to known species. Members of the genus can cause infection in humans and a wide range of domestic animals with different degrees of severity. Long-term persistence which, in some cases, is manifested as cyclic bacteremia has been demonstrated for several Anaplasma species. Zoonotic potential has been shown for A. phagocytophilum, the agent of human granulocytic anaplasmosis, and for some other Anaplasma spp. that suggests a broader medical relevance of this genus. Genetic diversity of Anaplasma spp. has been intensively studied in recent years, and it has been shown that some Anaplasma spp. can be considered as a complex of genetically distinct lineages differing by geography, vectors, and host tropism. The aim of this review was to summarize the current knowledge concerning the natural history, pathogenic properties, and genetic diversity of Anaplasma spp. and some unclassified genovariants with particular attention to their genetic characteristics. The high genetic variability of Anaplasma spp. prompted us to conduct a detailed phylogenetic analysis for different Anaplasma species and unclassified genovariants, which were included in this review. The genotyping of unclassified genovariants has led to the identification of at least four distinct clades that might be considered in future as new candidate species.
Article
Red foxes (Vulpes vulpes) have been recognized to harbour and transmit a wide range of tick-borne pathogens (TBPs) including those of zoonotic concern. To investigate the prevalence and the distribution of TBPs and of Leishmania infantum in foxes (n = 244), spleen samples were collected within the frame of a multi-regional wildlife health surveillance program in Italy. A combined PCR/sequencing approach was performed for the detection of Anaplasma spp., Babesia spp., Borrelia spp., Ehrlichia spp., Hepatozoon spp. and L. infantum DNA. Overall, 146 foxes (59.8%, 95% CI: 53.6-65.8) tested positive for at least one pathogen with Hepatozoon canis being the most prevalent (i.e., n = 124; 50.8%, 95% CI: 44.6-57.0), followed by Babesia vulpes (n = 20; 8.2%, 95% CI: 5.4-12.3), different spirochete species from Borrelia burgdorferi sensu lato complex (n = 9; 3.7%, 95% CI: 1.9-6.9), Ehrlichia canis and L. infantum (n = 7; 2.9% each, 95% CI: 1.4-5.8), Anaplasma platys (n = 4; 1.6%, 95% CI: 0.6-4.1), Anaplasma phagocytophilum ecotype I and Candidatus Neoehrlichia sp. (n = 3; 1.2% each, 95% CI: 0.4-3.5). All samples scored negative for Babesia canis and Borrelia miyamotoi. This study revealed the presence of spirochetes from B. burgdorferi s.l. complex, Ca. Neoehrlichia sp., A. platys and A. phagocytophilum ecotype I in red fox population from Italy, underling the necessity to monitoring these carnivores, mainly because they live in contact with dogs and humans. Data on the tick fauna circulating on wildlife species will complement information herein obtained, instrumentally to establish preventive strategies for minimizing the risk of infection for animals and humans.