Parasitology International 84 (2021) 102410

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Parasitology International
journal homepage: www.elsevier.com/locate/parint

Vector-borne pathogens in clinically healthy military working dogs in

eastern Austria
Bernhard W. Sonnberger a, Barbara Graf a, Reinhard K. Straubinger b, Dietmar Rackl c,
Adelheid G. Obwaller d, Roman Peschke a, Bita Shahi Barogh a, Anja Joachim a,
Hans-Peter Fuehrer a, *
a
Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria
b
Chair for Bacteriology and Mycology, Institute for Infectious Diseases and Zoonoses, Faculty of Veterinary Medicine, LMU Munich, Germany
c
Federal Ministry of Defense, Joint Support Command, Veterinary Service, Vienna, Austria
d
Federal Ministry of Defense, Division of Science, Research and Development, Vienna, Austria

A R T I C L E I N F O A B S T R A C T

Keywords: Military working dogs have an increased risk of acquiring an infection with vector-borne pathogens due to kennel
Canine vector-borne diseases housing and regular exposure to wildlife and vectors. To evaluate the level of infections in clinically healthy dogs
Babesia canis of the Austrian Armed Forces, 94 individuals of the Military Working Dog Training Centre (MWDTC) Kaiser­
Borrelia burgdorferi
steinbruch/eastern Austria were examined in August 2016, February 2019 and August 2019. A modified Knott
Dirofilaria repens
Military working dogs
test was used to determine the presence of microfilariae, PCR for DNA detection of filarioid nematodes (incl.
Dirofilaria), Leishmania spp., piroplasms, Borrelia spp., Bartonella spp. and Anaplasmataceae, and serological
examination for antibodies against Borrelia burgdoferi s. l. and Leishmania infantum in all dogs. Two dogs were
positive for Dirofilaria repens in the Knott test, and one of them also by PCR. Six clinically healthy dogs (4.2%)
were positive for Babesia canis (PCR). In serology, 10 (10.6%) of the dogs were positive for specific antibodies
against Borrelia burgdoferi s. l. The results suggest that the current measures against arthropod vector exposure
and the pathogens they can transmit are not fully sufficient for these dogs. Further investigations of the tick and
mosquito fauna in this area will shed more light on the risk of exposure for both the dogs and the staff of the
MWDTC.

1. Introduction infections with vector-borne organisms. Military working dogs spend a

larger part of their lives outdoors and also train and work in areas that
Vector-borne diseases are of great relevance to both human and pet dogs rarely visit due to restricted access for non-military persons and
animal health in tropical, but also in temperate climates, including animals. Therefore, their exposure to vectors in such a “small wilder­
central Europe. Filarioid helminths of veterinary and/or medical rele­ ness” must be considered higher than that for dogs in (peri-)urban areas.
vance are endemic in Mediterranean and eastern European regions, but Similar to hunting dogs, they also reflect the exposure of wildlife to
an increasing number of occurrences of these pathogens are also re­ vectors and the pathogens they harbour. As recent studies show, work­
ported from central Europe [1–3]. Tick-borne diseases of dogs, such as ing dogs that move a lot in the wild together with their trainers can also
babesiosis and anaplasmosis, are endemic to Austria [4]. The incidence be suitable sentinels for human zoonotic vector-borne diseases as well
and frequency of vector-borne pathogens are directly related to the [6].
presence of vectors and the possibility of an encounter between the The present study was conducted (a) to evaluate the presence of
vector and a potential host [5]. Exposed persons and animals that spend vector-borne infections in military dogs in eastern Austria and (b) to add
much time outdoors in vector habitats could therefore have an increased knowledge on the ecological networks of vector-borne diseases and
risk of contracting arthropod vectors and consequently of acquiring potential pathogen spill-overs to domestic animals and humans in the

* Corresponding author.
E-mail addresses: R.Straubinger@lmu.de (R.K. Straubinger), dietmar.rackl@bmlv.gv.at (D. Rackl), adelheid.obwaller@bmlv.gv.at (A.G. Obwaller), Roman.
peschke@vetmeduni.ac.at (R. Peschke), Bita.ShahiBarogh@vetmeduni.ac.at (B. Shahi Barogh), Anja.Joachim@vetmeduni.ac.at (A. Joachim), Hans-Peter.
Fuehrer@vetmeduni.ac.at (H.-P. Fuehrer).

https://doi.org/10.1016/j.parint.2021.102410
Received 29 April 2021; Received in revised form 1 June 2021; Accepted 17 June 2021
Available online 22 June 2021
1383-5769/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
B.W. Sonnberger et al. Parasitology International 84 (2021) 102410

particular ecological setting of dogs in areas with restricted human ac­ days, because they were not always available at the centre at the time of
tivities. The dog population of the present study is located in an area in blood collection; some had left and new animals had joined or were
eastern Austria known to be endemic for various tick and mosquito born. In the MWDTC’s own Rottweiler breeding unit, the annual
species. To evaluate the presence of vector-borne pathogens in the dog numbers of births varied; usually one or two litters were borne each
population studied, blood samples from healthy military working dogs year. A single sample was obtained from 56.4% of the individuals, two
were examined for the presence of Leishmania infantum, Babesia spp., samples were collected from 34.0% and three samples from 9.6% of the
Hepatozoon canis, Dirofilaria spp., Borrelia spp., Bartonella spp. and dogs.
Anaplasmataceae. In total, 144 blood samples from the 94 dogs were available for PCR
testing. Borrelia burgdorferi s. l.-specific serology and Leishmania spp.-
2. Materials and methods specific serology were performed once for all dogs or for 88 of the
dogs (all except the youngest puppies born in 2019), respectively.
2.1. Animals and blood samples Ethical approval for this study was obtained from the Ethics Com­
mittee of the University of Veterinary Medicine Vienna.
In total, 94 clinically healthy animals at the Military Working Dog
Training Centre in Kaisersteinbruch (MWDTC), Burgenland (eastern 2.2. Modified Knott test
Austria), were included in this study. The most common breeds (64.9%)
were Rottweilers, followed by Labrador Retrievers (9.6%), Malinois For the detection of Dirofilaria spp. microfilariae, a modified Knott
(8.5%) and German Shepherds (5.3%). One dog each was a Tervueren, a test [7]. was performed with all blood sample (n = 144).
Chihuahua and a Prager Rattler, for the rest (8.5%) the breed was un­
known. More than half of the dogs (55.3%) were born at the MWDTC 2.3. Serology
within the centre’s own breeding program, and according to the centre’s
documentation system, 23.4% of the tested dogs were imported from In total 94 blood sera of the military working dogs were tested once
other countries in Europe (Table 1). The gender distribution was for Borrelia burgdorferi s. l. -specific antibodies by using a kinetic ELISA
balanced (51.1% males and 48.9% females). The majority of the dogs (KELA) [8] and a commercially available line immunoblot assay (LIA,
was between 6 months and 3 years old at the time of sampling (Fig. 1). Virotech, Rüsselsheim, Germany) [9], detecting IgG antibodies. Samples
None of the dogs had a history of travel (after acquisition) or mission to were also tested with an in-house indirect immunofluorescence antibody
other countries. test (IFAT) for the detection of Leishmania infantum-specific antibodies
The military working dogs were kept in kennels and treated monthly (IgG). For this, serial serum dilutions (1,20 to 1360) were prepared with
against gastrointestinal helminths (combination therapy with febantel, PBS. Subsequently, antigen-coated slides (prepared from L. infantum
praziquantel and pyrantel; dose according to body weight). They were grown in vitro in RPMI 1640 supplemented with HEPES, sodium bi­
vaccinated against canine distemper virus, kennel cough (adenovirus carbonate, 10% FCS and penicillin/streptomycin at 28 ◦ C under 5%
type 2 and parainfluenza), hepatitis contagiosa canis, parvovirosis, CO2) were transferred to a humidity chamber and each field was covered
leptospirosis and rabies. with 25 μl of diluted serum. The slides were then incubated at 37 ◦ C for
The general management against ectoparasites consisted of spot-on 30 min, washed twice in PBS (2 × 7 min) and incubated for another 30
application of permethrin (Exspot®, MSD, Vienna, Austria) until 2017, min at 37 ◦ C with secondary antibody (FITC-conjugated anti-dog-IgG,
and after that, of oral application of fluralaner (Bravecto®; MSD, The Binding Site, Birmingham, UK), diluted 1:30 and mixed with a
Vienna, Austria) every three months. Dogs deployed in military missions drop of Evans Blue solution to reduce background fluorescence. The
abroad received an additional selamectin treatment as a spot-on before slides were then covered with PBS-glycerine and a cover slip, and
leaving (Stronghold®, Zoetis, Vienna, Austria). examined by fluorescence microscopy at 500× magnification under
Apart from episodic military missions across Austria, when off duty, immersion oil. Positive and negative control sera were prepared in
the enrolled dogs were kennelled in their handler’s residence, or in in­ parallel.
door or outdoor kennels until they were assigned to a military dog
handler at the MWDTC, and spent their complete life in the MWDTC. 2.4. Molecular analyses
Five of the tested dogs were privately owned by the employees at the
MWDTC, but also spent much time at the MWDTC. DNA was extracted from whole blood and peripheral blood mono­
Blood (EDTA and serum) samples were collected in August 2016, nuclear cells (PBMCs) using the erythrocyte lysis buffer of the High Pure
February 2019 and August 2019 from the Vena saphena lateralis or the PCR Template Preparation Kit (Roche, Mannheim, Germany) according
Vena cephalica, and transferred to the University of Veterinary Medicine to the manufacturer’s instructions. PCRs were conducted in an Eppen­
for further analysis. The dogs were clinically examined before sampling, dorf Mastercycler® Pro, Eppendorf, Hamburg, Germany to screen for
and the handlers were interviewed for the medical history of the dogs. DNA of filarioid helminths, Leishmania spp., Anaplasmataceae, Barto­
Being a dynamic population, not all dogs could be sampled at specified nella spp., Borrelia burgdorferi s.l. and piroplasms (Table 2).
PCR products were separated by electrophoresis on 2% agarose gels
stained with Midori Green Advance (Nippon Genetics Europe, Düren,
Table 1 Germany). Finally, purified PCR products were sequenced by LGC Ge­
Origin of the included dogs (n = 94). nomics GmbH (Berlin, Germany), and sequences were compared for
Origin Number of dogs [%] similarity using the Basic Local Alignment Search Tool (BLAST) in
Austria – military centre 52 [55.3]
GenBank® (http://www.ncbi.nlm.nih.gov/BLAST).
Austria - elsewhere 20 [21.3]
Germany 4 [4.3] 3. Results
Hungary 2 [2.1]
Netherlands 2 [2.1]
All dogs appeared clinically healthy during clinical examinations and
Slovenia 2 [2.1]
France 1 [1.1] blood collection. Individual dogs had histories of performance-
Croatia 1 [1.1] orthopaedic issues. However, clinical signs were limited to individuals
Czech Republic 1 [1.1] and did not extend over longer time periods. There were no reports of
Greece 1 [1.1] reduced performance, loss of body mass, circulation problems or other
Unkown 8 [8.5]
abnormalities reported by the trainers.

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B.W. Sonnberger et al. Parasitology International 84 (2021) 102410

Fig. 1. Age of the dogs at the time of blood sampling.

Table 2
PCR protocols for molecular analyses.
Organism Locus Primer sequences Amplification protocol Product size Reference
[bp]

Leishmania SSU LEI_70L_for: 5´-CGCAACCTCGGTTCGGTGTG-3′ 94 ◦ C 2 min 345 bp SPANAKOS et al.,

rRNA LE_70R_rev: 5´-CGCGGTGCTGGACACAGGGTA-3´ 40×: 94 ◦ C 1 min, 65 ◦ C 90 s, 72 ◦ C 90 s 2002
72 ◦ C 1 0 min
Anaplasmataceae 16S EHR16SD_for: 5´-GGTACCYACAGAAGAAGTCC-3´ 95 ◦ C 2 min 345 bp PAROLA et al., 2000
rRNA EHR16SR_rev: 5´-TAGCACATCATCGTTTACAGC-3 35×: 94 ◦ C 1 min, 54 ◦ C 30 s, 72 ◦ C 30 s
72 ◦ C 5 min
Filarioid COI H14FilaCOIRw: 5´-GCCTATTTTGATTGGTGGTTTTGG- 95 ◦ C 2 min 724 bp HODŽIĆ et al., 2015
nematodes 3 35×: 95 ◦ C 1 min, 53 ◦ C 1 min, 72 ◦ C 1
H14FilaCOIRv: 5´- min
AGCAATAATCATAGTAGCAGCACTAA-3´ 72 ◦ C 5 min
Piroplasms 18S Nest 1: Nest 1: Nest 1: 700 bp ZINTL et al., 2011
rRNA BTH–1F: 5´- 94 ◦ C 2 min
CCTGAGAAACGGCTACCACATCT-3´ 40×: 95 ◦ C 30 s, 68 ◦ C 1 min, 72 ◦ C 1 est 2: 561 bp
min
BTH-1R: 5´-TTGCGACCATACTCCCCCCA-3´ 72 ◦ C 10 min
Nest 2:
G-2_for: 5´-GTCTTGTAATTGGAATGATGG-3′ Nest 2:
94 ◦ C 2 min
40×: 95 ◦ C 30 s, 60 ◦ C 1 min, 72 ◦ C 1
G-2_rev: 5´-CCAAAGACTTTGATTTCTCTC-3´ min
72 ◦ C 10 min
Borrelia 16S Borr_allg_for: 5´-ACGCTGGCAGTGCGTCTTAA-3´ 94 ◦ C 5 min 674 bp LIEBISCH et al., 1998
rRNA Borr_allg_rev: 5´-CTGATATCAACAGATTCCACCC-3´ 40×: 94 ◦ C 1.5 min, 63 ◦ C 2 min, 72 ◦ C 2
min
72 ◦ C 10 min
Bartonella gltA BhCS.871p: 5´-GGGGACCAGCTCATGGTGG-3´ 94 ◦ C 5 min 379 bp NORMAN et al.,
BhCS.1137n: 5´-AATGCAAAAAGAACAGTAAACA-3´ 40×: 94 ◦ C 1 min, 54 ◦ C 1 min, 72 ◦ C 1 1995
min
72 ◦ C 10 min

Microfilariae were detected by the Knott test in two samples, both two Malinois) were from Austria, and the three Rottweilers were born in
from 2016, resulting in a relative prevalence of 1.4%. These were the MWDTC (Table 3). The Malinois, which was positive for D. repens,
identified morphologically as Dirofilaria repens. One of the positive dogs was also positive for B. canis. Of the B. canis DNA sequences, five samples
was a male Rottweiler born in Hungary and the other one a Malinois were 100% and one 99% similar to isolates found in Dermacentor retic­
from Austria. Only the sample from the latter dog could be confirmed as ulatus and in foxes in Austria (KY693669.1, KY447296.1) [10]. The PCR
D. repens by sequencing after PCR. Babesia canis DNA was detected in six for other pathogens (Anaplasmataceae and Bartonella spp.) were all
samples (4.2%) from 2016 (Table 3). None of these positive dogs had negative.
had any missions abroad. Except for one Malinois from Hungary, the None of the animals was positive for Leishmania spp.-specific anti­
B. canis-positive dogs (three Rottweilers, one Tervueren and a further bodies in serology, but ten individuals (10.6%) were classified as

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B.W. Sonnberger et al. Parasitology International 84 (2021) 102410

Table 3 Previous reports also indicated that B. canis is endemic in eastern Austria
Dogs positive for Babesia canis and Dirofilaria repens by PCR and amplicon [16,17]. The prevalence of B. canis of 6.4% in the present study can be
sequencing. MWDTC: Military Working Dog Training Centre. considered low to moderate compared to other European countries [18].
Breed Age at Origin Pathogens Accession nos. Different studies showed variable prevalences. Sled dogs also kept under
sampling detected outdoor conditions in Poland showed a much higher prevalence for
[years] B. canis with 25.3% positivity in 82 dogs tested by PCR over a 2-year
Rottweiler 1 MWDTC Babesia canis MW588421 period [19]. A study from 2013 showed a seroprevalence of 12.8% in
Tervueren 3 Austria Babesia canis MW588421 90 dogs in eastern Austria [17]. Military dogs in extensive husbandry in
Malinois 6 Hungary Babesia canis MW588420
Portugal (n = 100) had a seroprevalence of 3% [20]. Seropositive dogs
Malinois 4 Austria Babesia canis/ MW588421/
Dirofilaria repens MW590257 (19.8% of 197 tested dogs) without any clinical signs were found in a
Rottweiler 11 MWDTC Babesia canis MW588421 study in Romania [21].
Rottweiler 0.5 MWDTC Babesia canis MW588421 No dog in the present study showed clinical signs indicative of
babesiosis, despite parasitaemia. Clinically inapparent infections with
B. canis have been reported previously [19,22]. In addition to the risk to
positive for Borrelia burgdorferi s. l.-specific antibodies either in the KELA
the infected individual to develop a clinically apparent babesiosis
or in the line immunoassay (Table 4). Again, the Malinois, which was
(which can take a fatal course in untreated dogs; Köster et al., 2015), the
already positive for D. repens and B. canis, was also positive for Borrelia
potential of parasite transmission by clinically healthy dogs (either to
burgdorferi s. l.-specific antibodies.
the vector tick or to other dogs by blood transfusion) should not be
neglected. Infected dogs are therefore a risk factor for the spread of
4. Discussion
B. canis in Austria and neighbouring countries.

4.1. Dirofilaria repens

4.3. Borrelia burgdorferi sensu lato
In the modified Knott test microfilariae of D. repens were detected in
two out of 94 dogs. Only one of those blood samples produced a positive No dog was positive for B. burgdorferi s. l. when tested with PCR, but
result in the PCR, and sequence analysis confirmed the morphological 10.6% were positive for specific antibodies against B. burgdorferi s.l.,
identification of D. repens. This filarioid species is known to be endemic indicating previous tick exposure and infections. A determination of the
in Austria and has been detected in dogs with and without a history of seroprevalence of B. burgdorferi s. l. from 90 dogs from the same area in
travel to other endemic countries [1,11], as well as in the Anopheles Austria using a different method showed positive results in 31.1% of the
maculipennis complex, An. algeriensis, and An. plumbeus from eastern dogs [4]. In Germany, a prevalence of 6.9% for antibodies against
Austria [2,12]. Since one of the two positive dogs in the present study B. burgdorferi s.l. in dogs was reported [23]. In Hungary, a country
was born in Hungary, a known endemic country for D. repens [13], it bordering eastern Austria, healthy dogs were examined and 0.4% were
could not be unequivocally determined whether this specific infection seropositive for B. burgdorferi s. l. [24]. Thus, the seroprevalence
was of autochthonous origin; however, the infected Malinois was born determined in the present study may be considered moderate compared
and bred in Austria and had no known travel history; consequently, an to other studies.
autochthonous infection is assumed for this case. The local transmission The distribution of B. burgdorferi s. l. is strictly linked to the distri­
of D. repens is associated with two conditions; the presence of competent bution of its main vector, Ixodes ricinus [25]. In the province of Styria, in
mosquito vector species, which are present in Austria [2], and the south-eastern Austria, the prevalence of B. burgdorferi s. l. in I. ricinus
presence of a minimum number of dogs with circulating microfilariae in ticks collected from 2002 to 2003 was 25.7% [26]. It can be assumed
the blood [14]. The MWDTC and adjacent area would fulfil both con­ that military working dogs experience relatively high risks of contract­
ditions. The dogs of the Centre are housed in kennels, spending most of ing Borrelia spp.-infections due to the exposure to the tick vector
their lives outdoors and exposed to mosquito vectors. No D. immitis I. ricinus, which is the most abundant tick species in eastern Austria, and
antigen test was performed and thus an infection with macrofilariae of is known for its wide pan-European distribution [15,27,28].
D. immitis only might have been missed. Studies from Germany show a similarly high prevalence of
B. burgdorfer s. l. in dogs and humans [29,30]. These results imply that
4.2. Babesia canis the military dog handlers may experience a similar infection risk (and
infection prevalence) as their dogs. Additionally, another pathogenic
DNA of Babesia canis was detected in six of the 94 dogs. Five animals Borrelia species, B. miyamotoi, has recently been reported from Austria,
originated from Austria and one from Hungary. The main vector for this with one case of associated relapsing fever in a patient [31,32].
parasite, Dermacentor reticulatus, is known to be endemic in eastern
Austria. The MWDTC is located in an area where D. reticulatus infections 5. Conclusion and outlook
in dogs have been detected previously [15], and the presence of B. canis
in D. reticulatus later confirmed endemicity for this pathogen [10]. In the present study, dogs exposed to mosquitoes and ticks in their

Table 4
Dogs serologically positive for Borrelia.
Breed Age at sampling [years] Origin KELA (units) VlsE0Mix dog OspA0Mix DbpA0Mix OspC0Mix 39 kDa (BmpA) 58 kDa 83 kDa

Rottweiler 6 MWDTC 347.7 3 0 0 0 2 2 3

Rottweiler 10 France 116.8 1 0 0 0.5 0 0.5 0
Rottweiler 3 MWDTC 309.7 2 0 0 0 0.5 1 3
Malinois 4 Austria 131.4 1 0 0 0 0.5 1 0
Rottweiler 1 MWDTC 117.8 2 0 0 1 0 0 0
Rottweiler 3 MWDTC 114.5 1 0 0 0 0 0 2
Malinois unknown Unknown 110.8 1 0 0 0 0.5 0 2
unkown unkown Unknown 122.6 1 0 0 0 0.5 0 2
Rottweiler 2 MWDTC 97.2 2 0 0 1 0 0.5 0
Rottweiler 1 MWDTC 175.7 2 0 0 0.5 0 0 0

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