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Background Vector-borne pathogens (VBPs) are increasing in significance in veterinary medicine and public health settings, with wildlife playing a potentially crucial role in their transmission. Eurasian badgers (Meles meles) are widely distributed across Europe. However, information currently available on the prevalence of VBPs in badgers is limited. The objective of the current study was to investigate the occurrence of Anaplasmataceae, Bartonella spp., Mycoplasma spp., Rickettsia spp., Piroplasmida, Trypanosomatida and Filarioidea in badgers and subsequently, based on the results, assess the potential risk to domestic animals, other wildlife and humans. Methods Between 2017 and 2021, blood or spleen samples from 220 badgers were collected in nine continental European countries: Austria (n = 7), Bosnia and Herzegovina (n = 2), Croatia (n = 22), France (n = 44), Germany (n = 16), Hungary (n = 7), Italy (n = 16), Romania (n = 80) and Serbia (n = 26). VBPs were identified by performing PCR analysis on the samples, followed by Sanger sequencing. Additionally, to distinguish between different Babesia lineages we performed restriction fragment length polymorphism (RFLP) analysis on piroplasm-positive samples, using HinfI as restriction enzyme. A phylogenetic analysis was performed on Mycoplasma spp. Results The pathogens identified were Babesia sp. badger type A (54%), B (23%), and C (37%); Trypanosoma pestanai (56%); Mycoplasma sp. (34%); Candidatus Mycoplasma haematomelis (8%); Candidatus Mycoplasma haematominutum (0.5%); and Ehrlichia spp. (2%). Rickettsia spp., Bartonella spp. and filarioid nematodes were not detected among the tested samples. Conclusions The large sample size and diverse study populations in this study provide valuable insights into the distribution and epidemiology of the analyzed pathogens. Some of the VBPs identified in our study show high similarity to those found in domestic animals, such as dogs. This finding suggests that badgers, as potential reservoirs for these pathogens, may pose a threat not only to other wildlife but also to domestic animals in close vicinity. Continuous surveillance is essential to monitor VBPs in wildlife as a means to enable the assessment of their impact on other wildlife species, domestic animals and human health. Graphical Abstract
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Lindhorstetal. Parasites & Vectors (2024) 17:451
https://doi.org/10.1186/s13071-024-06515-y
RESEARCH
Molecular analysis ofvector-borne
pathogens inEurasian badgers (Meles meles)
fromcontinental Europe
Zoë Tess Lara Lindhorst1† , Sebastian Brandstetter1†, Maria Sophia Unterköfler1 , Barbara Eigner1 ,
Joachim Spergser2 , Marc Colyn3, Peter Steinbach4,5 , Duško Ćirović6 , Nikica Šprem7 , Tomislav Dumić8 ,
Vincenzo Veneziano9 , Franz Müller10^, Josef Harl11,12 , Georgiana Deak13 , Angela Monica Ionică14 ,
Mike Heddergott4 and Hans‑Peter Fuehrer1*
Abstract
Background Vector‑borne pathogens (VBPs) are increasing in significance in veterinary medicine and public health
settings, with wildlife playing a potentially crucial role in their transmission. Eurasian badgers (Meles meles) are widely
distributed across Europe. However, information currently available on the prevalence of VBPs in badgers is limited.
The objective of the current study was to investigate the occurrence of Anaplasmataceae, Bartonella spp., Mycoplasma
spp., Rickettsia spp., Piroplasmida, Trypanosomatida and Filarioidea in badgers and subsequently, based on the results,
assess the potential risk to domestic animals, other wildlife and humans.
Methods Between 2017 and 2021, blood or spleen samples from 220 badgers were collected in nine continental
European countries: Austria (n = 7), Bosnia and Herzegovina (n = 2), Croatia (n = 22), France (n = 44), Germany (n = 16),
Hungary (n = 7), Italy (n = 16), Romania (n = 80) and Serbia (n = 26). VBPs were identified by performing PCR analysis
on the samples, followed by Sanger sequencing. Additionally, to distinguish between different Babesia lineages we
performed restriction fragment length polymorphism (RFLP) analysis on piroplasm‑positive samples, using HinfI
as restriction enzyme. A phylogenetic analysis was performed on Mycoplasma spp.
Results The pathogens identified were Babesia sp. badger type A (54%), B (23%), and C (37%); Trypanosoma pestanai
(56%); Mycoplasma sp. (34%); Candidatus Mycoplasma haematomelis (8%); Candidatus Mycoplasma haematom‑
inutum (0.5%); and Ehrlichia spp. (2%). Rickettsia spp., Bartonella spp. and filarioid nematodes were not detected
among the tested samples.
Conclusions The large sample size and diverse study populations in this study provide valuable insights into the dis‑
tribution and epidemiology of the analyzed pathogens. Some of the VBPs identified in our study show high similarity
to those found in domestic animals, such as dogs. This finding suggests that badgers, as potential reservoirs for these
pathogens, may pose a threat not only to other wildlife but also to domestic animals in close vicinity. Continuous
Open Access
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Parasites & Vectors
Zoë Tess Lara Lindhorst and Sebastian Brandstetter contributed equally to
this work.
^Franz Müller died during the preparation of this article.
*Correspondence:
Hans‑Peter Fuehrer
Hans‑Peter.Fuehrer@vetmeduni.ac.at
Full list of author information is available at the end of the article
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Page 2 of 15
Lindhorstetal. Parasites & Vectors (2024) 17:451
Background
Emerging infectious diseases have been increasingly rec-
ognized and deemed significant in Europe in recent years
[1]. e primary reasons for the increasing emergence
of vector-borne pathogens (VBPs) in European coun-
tries, which were previously not present or less preva-
lent, include globalization, urbanization, global trade,
increased travel of humans and domestic animals and
climatic changes [2, 3]. ese substantial changes have
led to the widespread expansion of VBPs and arthropods
acting as their vectors [4]. Wildlife serve as potential res-
ervoirs and may therefore play a crucial role in the trans-
mission of these pathogens. Due to the zoonotic potential
of many VBPs and their capability to infect domestic ani-
mals, VBPs can be highly important factors in both vet-
erinary and human medicine [5].
e Eurasian badger (Meles meles) is among the most
prevalent medium-sized carnivores throughout Europe
[6], where it mainly inhabits woodlands [7, 8]. Despite
their typically shy and elusive behavior towards humans,
badgers, along with other wildlife, are regularly found
near human settlements, influenced by factors such as
habitat loss and food availability [7, 9]. To date, limited
information is available on the prevalence of VBPs in
badgers, particularly in populations beyond the UK [10].
However, the close coexistence of humans and badgers,
with the latter possibly serving as reservoirs for numer-
ous pathogens [1114], could pose a significant threat
to the health of both humans and domestic animals.
For example, badgers are considered to be an important
source of bovine tuberculosis in the UK due to the pos-
sible transmission of Mycoplasma bovis to cattle [15].
Among the most important parasitic pathogens found
in badgers are members of the order Piroplasmida. For
example, badger-associated Babesia parasites, which are
suspected to belong to the Babesia microti group, have
been detected in several European countries. Babesia sp.
isolates badger type A, type B [1, 1625] and Babesia sp.
badger, which was labeled type C in one study [25], have
been identified. In addition to badgers, badger-associated
Babesia have also been described in other carnivores in
Europe [16, 19, 26]. Ixodid ticks act as the main vectors
of Babesia spp. [27]. Another parasite detected in badg-
ers is Trypanosoma (Megatrypanum) pestanai, a species
that was identified in badgers from the UK, France and
Italy [2830], and in a dog (Canis lupus familiaris) from
Germany [31]. e role of badger fleas (Paraceras melis)
as vectors for T. pestanai had been confirmed [32] when
T. pestanai was also detected in ixodid ticks for the first
time in Italy [29]. In recent years, Eurasian badgers have
been confirmed as new hosts for the filarioid nematodes
Dirofilaria immitis [33] and Dirofilaria repens [11, 34].
However, the role of the badger as a definite host and a
reservoir for D. immitis and D. repens remains unclear
and requires further investigation. e most important
bacterial VBPs in European carnivores include Myco-
plasma spp., Anaplasmataceae, Borrelia spp., Bartonella
spp. and Rickettsia spp. [5, 35, 36]. Hemotropic myco-
plasmas (hemoplasmas) have been described in Eura-
sian badgers [37] and a Japanese badger (Meles meles
anakuma) [38]. Infections with Anaplasma phago-
cytophilum, Ehrlichia sp. [1, 39, 40] and Candidatus
Neoehrlichia sp. [18, 41] have been identified in badgers
in different European countries including, for example,
Italy and the Netherlands. Other pathogens that were
detected in badgers are Bartonella spp. [42], Rickettsia
spp. [43], and Borrelia spp. [13, 44].
e objective of our research was to investigate the
occurrence of Anaplasmatacea, Bartonella spp., Myco-
plasma spp., Rickettsia spp., Piroplasmida, Trypanoso-
matida and Filarioidea in badgers across nine European
countries. e findings of this study provide valuable
insights into the epidemiology of these pathogens in
badger populations, thereby aiding in the assessment of
potential cross-species transmission risks to domestic
animals, wildlife and humans.
Methods
Sample collection
Blood (n = 81) or spleen (n = 139) samples from 220 Eura-
sian badgers were analyzed for the presence of VBPs.
No animal was killed for the study; all samples were col-
lected from individuals that were found dead and were
taken from fresh cadavers (mainly between 24 and 48h
after death). e samples were collected from July 2017
to August 2021 in nine different European countries:
Austria (n = 7), Bosnia and Herzegovina (n = 2), Croa-
tia (n = 22), France (n = 44), Germany (n = 16), Hungary
(n = 7), Italy (n = 16), Romania (n = 80) and Serbia (n = 26)
(Fig.1). All samples were kept frozen at -20 °C in 70%
ethanol until the analysis at the University of Veterinary
Medicine, Vienna. During necropsy, all individuals were
examined to determine their sex and age, based on den-
tition stage and tooth wear [45]. According to the age
surveillance is essential to monitor VBPs in wildlife as a means to enable the assessment of their impact on other wild‑
life species, domestic animals and human health.
Keywords Vector‑borne pathogens, Badger, Babesia, Trypanosoma, Mycoplasma, Ehrlichia
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Page 3 of 15
Lindhorstetal. Parasites & Vectors (2024) 17:451
classification, the animals were then divided into two
groups: juvenile (aged 12 months) and adult (aged
> 12months).
DNA extraction, PCR amplication andsequencing
DNA was extracted from the samples using the DNeasy
Blood & Tissue kit (250) (QIAGEN, Hilden, Germany),
following the manufacturer’s instructions. Pathogen
detection was carried out following seven established
broad-range PCR assay protocols (Table 1; Additional
file 1: TableS1), with each protocol including positive
and negative controls. e PCR assays targeted sections
of the 16S ribosomal RNA (rRNA) in Mycoplasma spp.
and Anaplasmataceae, the 18S rRNA in Piroplasmida
and Trypanosomatida, the 16S-23S rRNA in Bartonella
spp., the 23S-5S rRNA intergenic spacer in Rickettsia spp.
and the COI gene in Filarioidea [4653]. PCRs were run
using GoTaq DNA Polymerase (Promega, Madison, WI,
USA).
e PCR products were subjected to electrophoresis
in 1.8% agarose gels with 4.2µl Midori Green Advance
DNA Stain (NIPPON Genetics EUROPE GmbH, Düren,
Germany). PCR products of positive samples were sent
to LGC Genomics (Berlin, Germany) for sequencing. e
chromatograms were visually inspected and edited (using
Chromas and GeneDoc) and compared against data from
the NCBI GenBank and the Barcode of Life (BOLD;
https:// www. bolds ystems. org/) databases.
In the case of Piroplasmida, a restriction fragment
length polymorphism method (RFLP), using a HinfI
restriction enzyme (Promega), was conducted on positive
samples to distinguish between Babesia lineages and to
detect mixed infections [48]. ree representative sam-
ples (one from each Babesia haplotype identified through
nested PCR) were selected for sequencing, and these
were then used as references for the remaining samples.
Phylogenetic analysis
One Mycoplasma sequence obtained in the present
study (GenBank accession no.: OQ749679) was used to
search for similar sequences using the BLAST function
on NCBI GenBank, setting the number of maximum
Fig. 1 Geographical distribution of the Eurasian badgers (Meles meles) examined in this study (n = 220). The size of the circles indicates the number
of samples per locality
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Lindhorstetal. Parasites & Vectors (2024) 17:451
target sequences to 5000. e filter was set to 82–100%
identity and 98–100% query coverage. e sequences
were aligned and sorted using the default option (FFT–
NS–2) in MAFFT v.7.520 [54]. All sequences featuring
obvious sequencing errors and ambiguous characters
were removed from the alignment and excluded from the
analysis.
To provide an overview of the diversity of haplotypes,
maximum likelihood (ML) and Bayesian inference (BI)
trees were calculated based on an alignment containing
392 sequences (1030 nucleotide positions). Gaps were
removed from the alignment using TrimAl v.1.3 (http://
phyle mon2. bioin fo. cipf. es/; [55]), and sequences were
collapsed to haplotypes using DAMBE v.7.3.32, leaving
237 haplotypes (963 nucleotide positions). e tree was
rooted with a sequence of Malacoplasma iowae (Gen-
Bank accession no.: CP129195).
An ML bootstrap consensus tree (1000 replicates) was
calculated using the W-IQ-TREE web server (http://
iqtree. cibiv. univie. ac. at/; [56]) applying the model
GTR + F + I + G4, which was suggested as the best fit for
the data set in the model test according to the Bayes-
ian inference criterion (BIC). e BI trees were calcu-
lated using MrBayes v.3.2.7 [57], applying the model
GTR + G + I. e analysis was run for 106 generations
(2 runs each with 4 chains), sampling every thousandth
tree. e first 25% of trees were discarded as burn-in
and a 50% majority-rule consensus tree was calculated
based on the remaining 750 trees. e ML and BI trees
were jointly created using the BI tree as a template, and
then graphically prepared indicating country and host
information in CorelDRAW 2024 (Corel, Ottawa, ON,
Canada).
Statistical analysis
Statistical analysis was conducted using R version 4.3.2 ®
Foundation for Statistical Computing, Vienna, Austria).
A Pearson’s Chi-squared (χ2) test was conducted to assess
the correlation between the detection of pathogens and
the sex and age of the animals. e association between
pathogen detection and country of origin, and month
and year of sample collection, respectively, was assessed
using Fisher’s exact test. Effects were considered statisti-
cally significant if P < 0.05.
Results
Pathogens detected in the 220 tested blood/spleen sam-
ples from badgers were Piroplasmida (195/220, 88.6%;
95% confidence interval [CI] 0.844–0.928), Trypano-
somatida (123/220, 55.9%; 95% CI 0.493–0.625), Myco-
plasma spp. (107/220, 48.6%; 95% CI 0.42–0.552) and
Anaplasmataceae (4/220, 1.8%; 95% CI 0.001–0.036).
Dirofilaria spp. and other filarioid helminths, Rickett-
sia spp. and Bartonella spp. were not detected in the
tested samples. No significant correlation was found
between pathogen occurrence and age (P = 0.219; odds
ratio [OR] Infinite, 95% CI 0.533-Infinity), sex (χ2 = 0.008,
df = 1, P = 0.928), year of sample collection (P = 0.439)
Table 1 Oligonucleotide sequences of primers used in the present study
COI Cytochrome c oxidase I, rRNA ribosomal RNA
Target organism (genetic
marker) Primer sequences (5 3) Product size (bp Annealing
temperature Reference
Mycoplasma spp.
(16S rRNA) HBT‑F: ATA CGG CCC ATA TTC CTA CG
HBT‑R: TGC TCC ACC ACT TGT TCA 600 60 °C [46]
Mycoplasma spp.
(16S rRNA) UNI_16 S_mycF: GGC CCA TAT TCC TAC GGG AAG CAG CAGT
UNI_16 S_mycR: TAG TTT GAC GGG GGG TGT ACA AGA CCTG 1000 56 °C [53]
Piroplasmida
(18S rRNA) BTF1: GGC TCA TTA CAA CAG TTA TAG
BTR1: CCC AAA GAC TTT GAT TTC TCTC 930 52 °C [48]
BTF2: CCG TGC TAA TTG TAG GGC TAA TAC
BTR2: GGA CTA CGA CGG TAT CTG ATCG 800 62 °C
Trypanosomatida
(18S rRNA) Tryp_18S_F1: GTG GAC TGC CAT GGC GTT GA
Tryp_18S_R1: CAG CTT GGA TCT CGT CCG TTGA ~ 1320 56 °C [51]
Tryp_18S_F2: CGA TGA GGC AGC GAA AAG AAA TAG AG
Tryp_18S_R2: GAC TGT AAC CTC AAA GCT TTC GCG 960 56 °C
Bartonella spp.
(16S23S rRNA) Bartgd_for: GAT GAT GAT CCC AAG CCT TC
B1623_rev: AAC CAA CTG AGC TAC AAG CC 179 60 °C [49]
Rickettsia spp.
(23S5S rRNA) Ricketts_ITS_for: GAT AGG TCG GGT GTG GAA G
Ricketts_ITS_rev: TCG GGA TGG GAT CGT GTG ~ 400 52 °C [52]
Anaplasmataceae
(16S rRNA) EHR16SD_for: GGT ACC YAC AGA AGA AGT CC
EHR16SR_rev: TAG CAC TCA TCG TTT ACA GC 345 54 °C [50]
Filarioidea
(COI gene) COIint‑F: TGA TTG GTG GTT TTG GTA A
COIint‑R: ATA AGT ACG AGT ATC AAT ATC 668 52.3 °C [47]
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Lindhorstetal. Parasites & Vectors (2024) 17:451
and month of sample collection (P = 0.817). ere was a
significant correlation between pathogen occurrence and
country of origin (P = 0.013). Additional statistical analy-
ses are presented in Additional file1: TableS2.
Of the 220 badger samples tested, 47 (21.4%; 95% CI
0.16–0.268) tested positive for one pathogen only, and
77 (35.0%; 95% CI 0.287–0.413) and 76 (34.6%; 95% CI
0.287–0.413) showed a co-infection with two and three
pathogens, respectively (Fig. 2). e highest prevalence
of pathogens was found in Austria, Bosnia and Herzego-
vina, Croatia, Germany and Hungary (each 100%), fol-
lowed by Romania (94%), Serbia (92%), France (84%) and
Italy (63%) (Fig.3).
Among the 195 samples positive for Piroplasmida,
119 (61.0%; 95% CI 0.542–0.679) could be assigned to
Babesia sp. badger type A (GenBank accession number:
KT223484), 50 (25.5%; 95% CI 0.12–0.318) to Babesia sp.
badger type B (GenBank accession number: KT223485)
and 82 (42.1%; 95% CI 0.351–0.49) to Babesia sp. badger
type C (GenBank accession number: MG799847). Of
these 195 Piroplasmida-positive badgers, 140 (71.8%; 95%
CI 0.655–0.781) showed an infection with one lineage
only, 54 (27.7%; 95% CI 0.214–0.34) showed an infec-
tion with two lineages and one (0.5%; 95% CI 0–0.015)
showed an infection with all three lineages simulta-
neously (Fig. 4). ree sequences, representative for
the three haplotypes found in the present study, were
uploaded to NCBI GenBank (accession nos.: PP621229–
PP621231) (Table2). e representative samples for type
A and type B showed 100% identity to Babesia sp. badger
type A and Babesia sp. badger type B, respectively, found
in badgers from Spain. e representative sample for
type C showed 100% identity to Babesia sp. badger “iso-
late Badger-1,” found in a badger from China (Table2).
e prevalence of badger-associated Babesia spp. was
100% in Bosnia and Herzegovina, Germany and Hungary,
followed by Serbia (92%), Romania (90%), France (84%),
Austria (71%) and Italy (63%) (Fig.3).
All 118 samples that tested positive for Trypanosoma-
tida were infected with T. pestanai. Of these, 82 (69.5%;
95% CI 0.612–0.778) of the sequences were 99.1–100%
identical to those of T. pestanai from a badger in
France (GenBank accession number: AJ009159), and 22
(18.6%; 95% CI 0.116–0.257) were 98.2–100% identical
Fig. 2 Co‑infection scheme of detected pathogens. Numbers represent counts of Eurasian badgers (Meles meles) with respective pathogens
detected. Percentages represent the proportion of positive badgers among all badgers tested (n = 220). A, Babesia sp.; B, hemotropic mycoplasmas;
C, Trypanosoma pestanai; D, Ehrlichia sp.
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Lindhorstetal. Parasites & Vectors (2024) 17:451
to T. pestanai detected in a badger in Italy (GenBank
accession number: MZ144610). ree representative
sequences were uploaded to NCBI GenBank under
the accession numbers PP595227–PP595229 (Table2).
e sequence quality of the remaining 14 samples was
poor and therefore could not definitely be assigned to
a species, although the sequences were similar to those
of T. pestanai. e prevalence of T. pestanai was high-
est in Bosnia and Herzegovina (100%), followed by Ger-
many (75%), Austria (71%), Croatia (68%), Romania
(58%), Hungary (57%), Serbia (54%), France (45%) and
Italy (31%) (Fig.3).
Fig. 3 Map of the Eurasian badger (Meles meles) samples used for this study, showing the prevalence by state in Europe. The bar charts show
the prevalence (in %) of hemotropic mycoplasmas, Ehrlichia sp., Trypanosoma pestanai, Babesia sp. badger type A, Babesia sp. badger type B
and Babesia sp. badger type C by study state
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Lindhorstetal. Parasites & Vectors (2024) 17:451
Fig. 4 Co‑infection scheme of detected Babesia sp. lineages. Numbers represent counts of Eurasian badgers (Meles meles) with the respective
haplotype(s) detected. Percentages represent the proportion of positive badgers among Piroplasmida‑positive badgers (N = 195). A, Babesia sp.
badger type A; B, Babesia sp. badger type B; C, Babesia sp. badger type C
Table 2 Sequencing results for Ehrlichia spp., Trypanosoma pestanai and Eurasian badger‑associated Babesia spp. and their closest
relationship
Accession
no. (this
study)
Haplotype (this
study) Country (this
study) Reference
haplotype Reference host Reference country Reference
accession no. Identity (in %)
PP595801 Uncultured Ehrli-
chia sp. Croatia Uncultured Ehrli-
chia sp. Canis lupus
familiaris
Hungary MH020203 100.0
PP595227 Trypanosoma
pestanai
Austria Trypanosoma
pestanai
Meles meles France AJ009159 100.0
PP595228 Trypanosoma
pestanai
Austria Trypanosoma
pestanai
Meles meles France AJ009159 100.0
PP595229 Trypanosoma
pestanai
Romania Trypanosoma
pestanai
Meles meles Italy MZ144610 98.7
PP621229 Babesia sp. badger
type A France Babesia sp. badger
type A Meles meles Spain KT223484 100.0
PP621230 Babesia sp. badger
type B Germany Babesia sp. badger
type B Meles meles Spain KT223485 100.0
PP621231 Babesia sp. badger
type C Croatia Babesia sp. badger Meles meles China MG799847 100.0
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Lindhorstetal. Parasites & Vectors (2024) 17:451
e sequences of three samples that tested positive
for Anaplasmataceae were 100% identical and one was
99.5% identical to Ehrlichia sp. found in a dog from Hun-
gary (GenBank accession number: MH020203). Due to
the low sequence qualities of two sequences, only one
was uploaded to GenBank under the accession number
PP595801 (Table 2). Ehrlichia sp. was found in Croa-
tia, Hungary and Romania (n = 2, 1 and 1, respectively)
(Fig.3).
Of the samples that tested positive for Mycoplasma
spp., 19 were subjected to additional PCRs (each with dif-
ferent primers amplifying larger PCR products), and the
resulting sequences were used for the phylogenetic anal-
ysis. Due to the low quality of the sequences, two were
excluded from the analysis. e remaining 17 sequences
were uploaded to NCBI GenBank under the acces-
sion numbers OQ749679–OQ749684 and OQ749687–
OQ749697) (Table3) and used for phylogenetic analysis
(Fig.5; Additional file2: Figure S1). Among the 107 sam-
ples that tested positive for Mycoplasma spp. during the
primary PCR, only two sequences showed 100% iden-
tity to sequences uploaded to GenBank, one (GenBank
accession number: OQ749681) to Mycoplasma sp. from a
European wild cat (Felis silvestris silvestris) in Bosnia and
Herzegovina (GenBank accession number: MF614158),
and one (GenBank accession number:OQ749683) to
Candidatus Mycoplasma haematomelis found in a
domestic cat (Felis catus) from Italy (GenBank accession
number: KR9055451). However, the results from both
samples differed from those obtained in the secondary
PCR, where they showed 98.8% identity to Mycoplasma
sp. found in an American mink (Neogale vison) from
Chile (GenBank accession number: MT462252), and
99.6% to Mycoplasma sp. found in a raccoon (Procyon
lotor) from the USA (GenBank accession number:
KF743733), respectively. e remaining 105 samples
showed identities of 96.04–99.8% (Table4) and notable
differences from those observed by the second PCR. e
prevalence of Mycoplasma spp. was highest in Hungary
(71%), followed by Austria (57%), Germany (56%), Roma-
nia (53%), Bosnia and Herzegovina (50%), Serbia (50%),
Croatia (45%), France (43%) and Italy (25%) (Fig.3).
Discussion
is study included a large (N = 220) number of exam-
ined badgers, originating from nine different countries in
Southern, Western and Eastern Europe [58], thereby pro-
viding a representative cross-section of the badger popu-
lation in continental Europe and its distribution [59]. It
should be noted, however, that the sample sizes varied
across the countries, ranging from two to 80 examined
samples (Fig.1).
e composition of pathogens identified in the samples
examined in the present study is roughly comparable to
that reported in other European studies. e prevalence
of T. pestanai was high (56%) in our study compared
to that reported in earlier studies where it ranged from
10% to 35% [28, 32, 60], but the majority of previous
publications reporting T. pestanai originated from the
UK [28, 32, 60]. Apart from these studies, in Europe, T.
pestanai has only been found in two badgers from Italy,
which already showed clinical symptoms prior to dying
[29]. e recent detection of badger-associated T. pesta-
nai in a dog from Germany suggests that the pathogen
can be transmitted to other carnivores [31]. erefore,
the high prevalence of this parasite in the present study
could represent a potential risk to both wild and domes-
tic carnivores, especially when their immune status is
Table 3 Sequencing results for Mycoplasma spp., using UNI_16 S_mycF and UNI_16 S_mycR primers, and their closest relationship,
according to GenBank BLAST results
Of the samples that tested positive for Mycoplasma spp., 19 were subjected to additional PCRs, of which 2 sequences of minor quality were excluded from the
phylogenetic analysis (n = 17)
Number
of
samples
Accession
number (this
study)
Country (this study) Reference haplotype Reference host Reference country Reference
accession number Identity (in %)
5 OQ749691 Germany, France Candidatus Myco
plasma haemato‑
melis
Meles meles anakuma Japan AB848713 99.7
1 OQ749696 Croatia Mycoplasma sp. Canis lupus familiaris Cambodia ON620261 99.4
1 OQ749681 Romania Mycoplasma sp. Neovison vison Chile MT462252 98.8
5 OQ749679 Romania Mycoplasma sp. Procyon lotor USA KF743733 99.6
2 OQ749689 Serbia, Croatia Mycoplasma sp. Procyon lotor USA KF743733 99.5
1 OQ749690 Germany Mycoplasma sp. Procyon lotor USA KF743733 99.4
1 OQ749687 Hungary Mycoplasma sp. Procyon lotor USA KF743733 99.4
1 OQ749688 Serbia Mycoplasma sp. Procyon lotor USA KF743733 99.3
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 9 of 15
Lindhorstetal. Parasites & Vectors (2024) 17:451
compromised [29]. Further research is needed in this
area to enable more precise conclusions.
In 2009, a new piroplasmid species was detected
in a badger in Spain (GenBank accession number:
FJ225390), which was considered to belong to Babe-
sia microti-like organisms [21] and which was later
found in several other studies and referred to as Babe-
sia sp. badger type A by some authors [1, 1619, 22,
25]. A different badger-associated Babesia species was
designated as type B [1, 17, 22, 25]. In 2021, another
Babesia species was found in one badger from the
Netherlands [25] which was > 99% identical to a spe-
cies found in a badger in an unpublished Chinese
study (GenBank accession number: MG799847). ese
authors did not upload it to the GenBank but referred
to it as Babesia sp. badger type C [25]. In the present
study, all three badger-associated Babesia species were
detected, with type A being the most prevalent (61%),
Fig. 5 Bayesian inference tree featuring 16S rRNA sequences (963 nucleotide positions) of selected Mycoplasma spp. Nodes are marked
with Bayesian posterior probabilities and maximum likelihood bootstrap values. Accession numbers, species name, host and country are provided
for every sequence. Sequences which are written in bold are from Meles meles, and sequences marked in red were obtained in this study. The scale
bar indicates the expected mean number of substitutions per site according to the model of sequence evolution applied. For reasons of clarity,
the number of sequences has been reduced to up to 4 sequences per clade. The full phylogenetic tree can be found in Additional file 2: Figure S1
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 10 of 15
Lindhorstetal. Parasites & Vectors (2024) 17:451
followed by type C (43%) and type B (25%). Interest-
ingly, Babesia sp. type C was not found in Austria,
France and Germany, but only in Italy and the coun-
tries of Eastern Europe (Fig.2). is geographical fea-
ture may be attributed to the natural barrier of the
Alps, which separates north-western and south-eastern
European countries, impacting wildlife and vectors.
e unexpectedly high prevalence of type C, previously
reported only in two other badgers from southern Italy
and China [16], may be attributed to the large sam-
ple size and diverse geographical origin of the badgers
examined, thus allowing for a more sensitive and dif-
ferentiated analysis. Moreover, previous studies rarely
examined samples from badgers south of the Alpine
and Apennine belts, which could also explain the very
low number of Babesia sp. type C found in past stud-
ies. Ixodid ticks are described as the primary vectors
for Babesia spp. in Europe, and some of these vectors
are prevalent in Alpine regions [61, 62]. While Babesia
sp. showing 100% identity to type C was found in Ixodes
canisuga, the fox tick, in Germany (GenBank accession
number: JX679177), Babesia sp. badger types A and
B have not yet been identified in ectoparasite vectors
[1, 1619, 22, 25]. However, the geographical distribu-
tion of type C could indicate, that this genotype needs
a different vector than types A and B. More studies are
needed to assess the vectors, definite hosts and the pre-
cise life cycle of this pathogen. Badger-associated Babe-
sia spp. have also been detected in wolves (Canis lupus)
[16], wildcats [26] and even dogs [19] in Europe. is
indicates that the pathogens are not strictly specific to
badgers and could therefore be a potential threat to
both wild and domestic carnivores.
In contrast to other studies carried out in Europe, A.
phagocytophilum was not detected in the present study
[1, 40]. ree sequences were 99.5–100% identical to
Ehrlichia sp. found in a dog from Hungary (GenBank
accession number: MH020203) [41].
To the authors’ knowledge, only two previous studies
reported hemoplasmas in badgers, with one describing
hemoplasmas in a Japanese badger [38] and the other
describing hemoplasmas in Eurasian badgers from Spain
[37]. e high prevalence in the present survey is in con-
cordance with the results reported from a study in Spain
[36], where the prevalence of hemoplasmas was 57%.
Similarly, a high diversity of Mycoplasma lineages was
observed in the present study (Table4).
To obtain more detailed genetic information, we
selected 19 Mycoplasma-positive samples for performing
an additional PCR in order to obtain a longer fragment
of the 16S rRNA gene. e aim was to draw conclusions
from the representative samples and to adapt the results
of the remaining 88 sequences accordingly. However, the
results differed significantly from one another, and DNA
fragments from samples with 100% identity showed high
deviations from results obtained with the primary PCR.
erefore, it was not possible to infer the Mycoplasma
species of the samples from the first PCR based on the
results of the representative 19 samples. e difference
in results indicates the presence of co-infections with
two or more Mycoplasma species in some of the badgers
examined.
e prevalence of hemoplasmas was highest in Hun-
gary, followed by Austria and Germany, and it was low-
est in Italy. Due to the sparse body of literature available
on hemoplasmas in badgers, a detailed comparison was
not possible. e prevalence of hemoplasmas in Euro-
pean wild carnivores showed variable results, ranging
from 2% in foxes (Vulpes vulpes) to 57% in badgers from
Spain [26, 37, 6365]. Although studies in domestic cats
Table 4 Sequencing results for Mycoplasma spp., using HBT‑F and HBT‑R primers, and their closest relationship, according to GenBank
BLAST results.
A Austria, B Bosnia and Herzegovina, C Croatia, F France, G Germany, H Hungary, I Italy, R Romania, S Serbia
Of the samples tested, 13 sequences of minor quality were excluded from further analysis (n = 94)
Number
of
samples
Country Reference haplotype Reference host Reference country Reference
accession number Identity (in %)
18 A, F, R, S Candidatus Mycoplasma haematomelis Meles meles anakuma Japan AB848713 99.3–99.7
1 R Candidatus Mycoplasma haematominu‑
tum Felis catus Italy KR905451 100.0
5 C, G, R Mycoplasma sp. Canis lupus familiaris Cambodia ON620261 99.1–99.8
11 F, H, R Mycoplasma sp. Procyon lotor USA KF743729 96.0–96.2
50 B, C, F, G,
H, I, R, S Mycoplasma sp. Procyon lotor USA KF743733 98.9–99.8
1 R Mycoplasma sp. Felis silvestris silvestris Bosnia and Herzegovina MF614158 100.0
8 G, H, R, S Mycoplasma sp. Felis silvestris silvestris Bosnia and Herzegovina MF614159 99.6–99.8
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 11 of 15
Lindhorstetal. Parasites & Vectors (2024) 17:451
and dogs revealed a higher prevalence of hemoplasmas in
warmer regions [46, 66], this difference does not appear
as pronounced among wild carnivores. In contrast, vari-
ations in prevalence are more noticeable across different
species. In our study, we observed a higher prevalence
in colder regions compared to Mediterranean countries
(e.g. Italy) (Fig. 3). is finding indicates that climatic
conditions may not be as crucial in the transmission of
hemoplasmas among wild carnivores as in pets. One pos-
sible explanation based on by new evidence is that hemo-
plasmas may not always require ectoparasite vectors, but
may be transmitted by different mechanisms, such as
fighting or social contact [67].
Leishmania infantum was previously identified in
badgers from Italy and Spain with a prevalence ranging
from 1.7% to 53% [1, 24, 6871]. In the present study,
however, despite examining large sample sizes from
Mediterranean countries where L. infantum is endemic
[72], none of the samples tested positive for this patho-
gen. Further studies on the prevalence of this parasite
in the Eurasian badger population in Europe, especially
in the Mediterranean region, could be of interest in the
future due to the badger’s risk of being potential reservoir
hosts of leishmaniasis [68].
Although we examined samples from countries
where Filarioidea, such as D. immitis and D. repens, are
described as endemic (e.g. Italy, Serbia, Hungary and
Romania) [7378], we did not detect any filarioid nema-
todes in our studies. Both D. immitits and D. repens have
been recently detected in badgers [11, 12, 33, 34], which
indicates that badgers are suitable hosts for Dirofilaria
spp. It should be considered that the prevalence of D.
repens in the two studies was quite low (10.6% in Rus-
sia and 1.9% in Poland), and to date, the presence of D.
immitis in badgers has only been reported from Romania
and Greece [12, 33].
Rickettsia spp. and Bartonella spp. were not detected
in this study. Notably, Rickettsia spp. are not regularly
tested for in badgers, and previous studies have primar-
ily detected these pathogens in skin biopsies rather than
spleen samples [20, 43, 79]. Regarding Bartonella spp.,
apart from one study from Spain that reported a preva-
lence of 12% [42], no other studies detected Bartonella
spp. in badgers, including those focusing on vectors
collected from badgers [20, 23, 80, 81]. Nevertheless, it
should be noted that the sample sizes used in the latter
four studies were small, ranging from three to 18 individ-
uals. Due to the infestation of the badgers with multiple
ectoparasites in the above-mentioned study from Spain
[42], a determination of vectors for Bartonella sp. found
in badgers is needed.
As wildlife-borne pathogens are responsible for > 70%
of emerging zoonotic infectious diseases [82] and also
play a crucial role as reservoirs for pathogens that can be
transmitted to domestic animals [20], studies on VBPs in
wildlife are becoming increasingly relevant. e badger-
associated Babesia spp., T. pestanai and Ehrlichia sp.
detected in this study are described as badger-specific
pathogens. However, badger-associated Babesia spp. and
Ehrlichia sp. as well as T. pestanai have been previously
found in dogs [19, 31, 41], therefore posing a potential
risk to pet dogs living in the vicinity of badgers. Hemo-
plasmas can infect a variety of mammals. e myco-
plasmas identified in this study were similar to those of
species detected in other wild and domestic carnivores.
Since hemoplasmas have also been described in humans
[8385], further investigation of the pathogens in wild
carnivores would be of interest.
Notably, the overall prevalence of pathogens was
comparatively low in France and, in particular, in Italy
(Fig.3), although no significant correlation was observed
between pathogen occurrence and the country of ori-
gin of the badgers. Given that many southern European
countries are known for various endemic VBPs and
their vectors [86, 87], it was anticipated that the preva-
lence of pathogens in these regions would be higher than
that in more northern parts of Europe. However, it is
important to note that much of the existing literature on
VBPs in these countries has primarily focused on filari-
oid nematodes and L. infantum, both of which were not
identified here [86, 87]. Regarding the low prevalence in
Italy, it should be mentioned that the majority of indi-
viduals tested in Italy came from the southern region of
Campania (n = 10), while the rest of the samples (n = 6)
originated from the northern regions Veneto and Friuli-
Venezia Giulia (Fig. 1). Although only four (40.0%) of
these 10 badgers from southern Italy tested positive, all
six (100.0%) badgers from the northern region tested
positive for at least one pathogen. As VBPs and their vec-
tors are quite widespread in southern Italian regions [88
90], this geographical distribution is rather surprising.
No statistically significant correlation was found
between the presence of pathogens and the sex or age of
the animals. Previous research examining the relation-
ship between the presence of VBPs and factors such as
sex or age has yielded contradictory results and shown
considerable variability depending on the specific patho-
gen under investigation [9196].
Conclusions
e large sample size and diverse study populations in
this research provide valuable insights into the distri-
bution and epidemiology of the pathogens analyzed in
Eurasian badgers. Several VBPs identified in the pre-
sent study are highly similar to those found in domestic
animals, such as dogs, which suggests that badgers may
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Page 12 of 15
Lindhorstetal. Parasites & Vectors (2024) 17:451
pose a threat to other wildlife and domestic animals in
their vicinity as potential reservoirs of these pathogens.
Continued surveillance is essential to monitor VBPs
in wildlife and to assess their impact on other wildlife,
domestic animals and human health.
Abbreviations
BI Bayesian inference
BIC Bayesian inference criterion
ML Maximum likelihood
RFLP Restriction fragment length polymorphism
VBP Vector‑borne pathogen
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13071‑ 024‑ 06515‑y.
Additional File 1: Table S1. PCR protocols and cycling conditions used in
the present study. Table S2. Positivity to one or more pathogens in badg‑
ers in continental Europe in relation to sex, age, country of origin, year of
sample collection, and month of sample collection.
Additional File 2: Figure S1. Bayesian Inference tree based on 16S rRNA
(963 nucleotide positions) sequences of selected Mycoplasma spp. Bayes‑
ian posterior probabilities and Maximum Likelihood bootstrap values are
provided for most nodes. Accession numbers, species name, host, and
country are provided for every sequence if available. Sequences that are
written in bold are from Eurasian badgers and sequences marked in red
were obtained in this study. The scale bar indicates the expected mean
number of substitutions per site according to the model of sequence
evolution applied.
Acknowledgements
We would like to thank the following people for providing sample material:
S Arnold, L Bohler, S Dietzel, M Dietrich, C Goeders, K Gunkel, M Hartmann, JL
Hammer, F Hummel, M Hoffmann, B König, F Röse, BP Zimmer and P Zimmer‑
mann. Research was supported by the Serbian Ministry of Education, Science
and Technological Development (451‑03‑65/2024‑03/ 200178).
Author contributions
MC, PS, DC, NS, TD, VV, FM, GD, AMI and MH: specification, sample collection
and preparation. ZTLL, SB, BE, JS, JH, GD, AMI and HPF: Pathogen screening
and molecular analysis. ZTLL, MH and HPF: writing of main manuscript text. All
authors read and approved the final manuscript.
Funding
This research received no external funding.
Availability of data and materials
The data presented in this study are contained within the article and supple‑
mentary material. Additional data can be provided on request.
Declarations
Ethics approval and consent to participate
According to national animal protection laws, only samples from animals
that died due to natural causes, external causes (e.g., roadkill) were examined.
As the project did not involve live animals or animal experiments, nor did it
involve sensitive patient data, the study does not have to be reported to the
Ethics and Animal Welfare Committee (ETK) according to point 1.2 and 1.3
of the guidelines regarding Good Scientific Practice (Ethics in Science and
Research) of the University of Veterinary Medicine Vienna.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1 Institute of Parasitology, Department of Biological Sciences and Pathobiol‑
ogy, University of Veterinary Medicine Vienna, Vienna, Austria. 2 Institute
of Microbiology, Department of Biological Sciences and Pathobiology,
University of Veterinary Medicine Vienna, Vienna, Austria. 3 UMR 6553 Ecobio,
Station Biologique, CNRS–Université de Rennes 1, Paimpont, France. 4 Musée
National d’Histoire Naturelle, Luxembourg, Luxembourg. 5 Faculty of Chem‑
istry, Georg‑August University of Göttingen, Göttingen, Germany. 6 Faculty
of Biology, University of Belgrade, Belgrade, Serbia. 7 Department of Fisheries,
Apiculture, Wildlife Management and Special Zoology, Faculty of Agriculture,
University of Zagreb, Zagreb, Croatia. 8 Department of Wildlife Management
and Nature Conservation, Karlovac University of Applied Sciences, Karlo‑
vac, Croatia. 9 Department of Veterinary Medicine and Animal Productions,
University of Naples Federico II, Naples, Italy. 10 Wildlife Biology Working Group,
Justus‑Liebig‑Universität Gießen, Giessen, Germany. 11 Institute of Pathology,
Department of Biological Sciences and Pathobiology, University of Veterinary
Medicine Vienna, Vienna, Austria. 12 Department of Experimental Pathology
and Laboratory Animal Pathology, Medical University Vienna, Vienna, Austria.
13 Department of Parasitology and Parasitic Diseases, University of Agricultural
Sciences and Veterinary Medicine of Cluj‑Napoca, Cluj‑Napoca, Romania.
14 Clinical Hospital of Infectious Diseases of Cluj‑Napoca, Cluj‑Napoca,
Romania.
Received: 16 July 2024 Accepted: 27 September 2024
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Background: Hippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligate blood-sucking ectoparasites of animals, and accidentally of humans. The potential role of hippoboscids as vectors of human and veterinary pathogens is being increasingly investigated, but the presence and distribution of infectious agents in louse flies is still unknown in parts of Europe. Here, we report the use of molecular genetics to detect and characterize vector-borne pathogens in hippoboscid flies infesting domestic and wild animals in Austria. Methods: Louse flies were collected from naturally infested cattle (n = 25), sheep (n = 3), and red deer (n = 12) across Austria between 2015 and 2019. Individual insects were morphologically identified to species level and subjected to DNA extraction for molecular pathogen screening and barcoding. Genomic DNA from each louse fly was screened for Borrelia spp., Bartonella spp., Trypanosomatida, Anaplasmataceae, Filarioidea and Piroplasmida. Obtained sequences of Trypanosomatida and Bartonella spp. were further characterized by phylogenetic and haplotype networking analyses. Results: A total of 282 hippoboscid flies corresponding to three species were identified: Hippobosca equina (n = 62) collected from cattle, Melophagus ovinus (n = 100) from sheep and Lipoptena cervi (n = 120) from red deer (Cervus elaphus). Molecular screening revealed pathogen DNA in 54.3% of hippoboscids, including infections with single (63.39%), two (30.71%) and up to three (5.90%) distinct pathogens in the same individual. Bartonella DNA was detected in 36.9% of the louse flies. Lipoptena cervi were infected with 10 distinct and previously unreported Bartonella sp. haplotypes, some closely associated with strains of zoonotic potential. DNA of trypanosomatids was identified in 34% of hippoboscids, including the first description of Trypanosoma sp. in H. equina. Anaplasmataceae DNA (Wolbachia spp.) was detected only in M. ovinus (16%), while < 1% of the louse flies were positive for Borrelia spp. and Filarioidea. All hippoboscids were negative for Piroplasmida. Conclusions: Molecular genetic screening confirmed the presence of several pathogens in hippoboscids infesting domestic and wild ruminants in Austria, including novel pathogen haplotypes of zoonotic potential (e.g. Bartonella spp.) and the first report of Trypanosoma sp. in H. equina, suggesting a potential role of this louse fly as vector of animal trypanosomatids. Experimental transmission studies and expanded monitoring of hippoboscid flies and hippoboscid-associated pathogens are warranted to clarify the competence of these ectoparasites as vectors of infectious agents in a One-Health context.
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