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ORIGINAL PAPER
Serological detection of Anaplasma phagocytophilum,
Borrelia burgdorferi sensu lato and Ehrlichia canis antibodies
and Dirofilaria immitis antigen in a countrywide survey
in dogs in Poland
Friederike Krämer &Roland Schaper &Bettina Schunack &Andrzej Połozowski &
Jolanta Piekarska &Aleksandra Szwedko &Robert Jodies &Dagmara Kowalska &
Dörte Schüpbach &Nikola Pantchev
Received: 14 May 2014 /Accepted: 6 June 2014
#The Author(s) 2014. This article is published with open access at Springerlink.com
Abstract Canine vector-borne diseases (CVBDs) have in-
creasingly become a focus of attention in the past few years.
Nevertheless, in many parts of Europe information on their
occurrence is still scarce. In a large study in Poland 3,094
serum samples taken from dogs throughout all 16 Polish
provinces were tested using a commercial kit for the detection
of circulating antibodies against Anaplasma phagocytophilum,
Borrelia burgdorferi sensulatoandEhrlichia canis and of
Dirofilaria immitis antigen. A total of 12.31 % (381/3,094;
95 % confidence interval [CI]: 11.18–13.52 %) and 3.75 %
(116/3,094; 95 % CI: 3.11–4.48 %) of the dogs were positive
for A. phagocytophilum and B. burgdorferi s.l. antibodies,
respectively. Furthermore, 0.26 % (8/3,094; 95 % CI: 0.11–
0.51 %) were positive for E. canis antibodies and 0.16 %
(5/3,094; 95 % CI: 0.05–0.38 %) for D. immitis antigen. The
highest percentages of A. phagocytophilum-positive dogs
were noted in Lesser Poland, Silesia and ŁódźProvinces.
For B. burgdorferi s.l., the highest prevalence was recorded
in ŁódźProvince. Co-infections with A. phagocytophilum
and B. burgdorferi s.l. were recorded in 1.71 % of all
examined dogs (53/3,094; 95 % CI: 1.29–2.23 %). One
dog even had a triple infection, testing positive for E. canis
too. Both A. phagocytophilum and B. burgdorferi s.l. have
previously been reported in Poland and were confirmed in
the present study by positive samples from all 16 provinces.
Concerning E. canis and D. immitis travel history or impor-
tation cannot be excluded as factors which may have deter-
mined the occurrence of these pathogens in the relevant
animals. Practitioners in Poland should be aware of the above
mentioned CVBDs and of prophylactic measures to protect
dogs and their owners.
Keywords Dog .Canine vector-borne diseases (CVBDs) .
SNAP® 4Dx® .Prevalence .Distribution .Poland
Introduction
Canine vector-borne diseases (CVBDs) have increasingly
become a focus of interest in recent years. Long-term climate
change on the one hand, and biotic factors —such as an
increase in reservoir abundance, changing habitat structure,
socio-political changes and, especially for dogs, increasing
F. Kr ä mer
Institute for Parasitology and Tropical Veterinary Medicine, Faculty
of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin,
Germany
R. Schaper (*):B. Schunack
Bayer Animal Health GmbH, 51373 Leverkusen, Germany
e-mail: Roland.Schaper@bayer.com
A. Połozowski :J. Piekarska
Department of Internal Medicine and Clinic of Horses, Dogs and
Cats, Faculty of Veterinary Medicine, Wroclaw University of
Environmental and Life Sciences, 50-366 Wroclaw, Poland
A. Szwedko
PPH Eskulap, 44-105 Gliwice, Poland
R. Jodies
Lekdijk Oost 11, 4112PB Beusichem, The Netherlands
D. Kowalska
Bayer Sp. z o.o., Animal Health Division, 02-326 Warsaw, Poland
D. Schüpbach:N. Pantchev
IDEXX Vet Med Labor GmbH, 71636 Ludwigsburg, Germany
Parasitol Res
DOI 10.1007/s00436-014-3985-7
travel and dog import for welfare reasons —on the other hand
are discussed in this context as parameters for the expansion of
vectors and pathogens into formerly unaffected areas. Two of
these canine vector-borne pathogens, Anaplasma
phagocytophilum and Borrelia burgdorferi sensu lato, have
now been reported in dogs in nearly all European countries. In
some countries, the pathogens have only been reported in the
vector, e.g., A. phagocytophilum in Ixodes ricinus ticks in
Finland (unpublished data by E. Hasu cited in Heikkilä et al.
2010), Estonia (Katargina et al. 2012) and Lithuania
(Paulauskas et al. 2012), or in animals other than dogs, e.g.,
in a cat in Finland (Heikkilä et al. 2010), but data on canine
prevalence of A. phagocytophilum have yet to be published.
From the Baltic States plus Belarus, for example, a canine
study with confirmed occurrence of A. phagocytophilum
could be found only for Latvia (Bērziņa and Matīse 2013).
Other studies screened only small canine populations in a
restricted focus.
The highest number of human cases of borreliosis in
Poland in 2011 was registered in Podlaskie Province with
75.5 per 100,000 people (Paradowska-Stankiewicz and
Chrześcijańska 2013). Most cases of borreliosis in Poland
originally occurred in this north-eastern region, but the
disease is no longer solely a problem in this part of the
country (Paradowska-Stankiewicz and Chrześcijańska
2013). Other studies on ticks and forest workers in the
north-western part of the country revealed prevalences
between 7.4 % (Skotarczak et al. 2002) and 16.7 %
(Skotarczak et al. 2003) in the tick population and 61 %
in forest workers (Niścigorska et al. 2003). Seropositivity
was also recorded in dogs in the north-western part of
Poland (Skotarczak and Wodecka 2003,2005). The main
vector in the area for the pathogen B. burgdorferi s.l. is
I. ricinus, which is generally distributed throughout the
country.
The pathogen A. phagocytophilum is reported to occur in
its vector I. ricinus in numerous studies in Poland. The prev-
alence in ticks has been reported across the country (from the
north-west (Rymaszewska 2005) to the south-east (Cisak et al.
2005)), ranging from 2.9 % in the central region (Warsaw)
(Zygner et al. 2008) to 76.7 % in the south (Lesser Poland)
(Asman et al. 2013). In man (mainly forest workers as an
especially tick-exposed group within the population), antibod-
ies against A. phagocytophilum have also been detected, e.g.,
in 17.7 % in north-eastern Poland (Roztocze National Park in
Lublin) (Cisak et al. 2005) and 19.8 % in the Lublin region
(Zwoliński et al. 2004). Finally, dogs have been screened in a
few studies, with 2/192 dogs being seropositive for
A. phagocytophilum in north-western Poland (Skotarczak
et al. 2004), 14 % of dogs suspected of having Lyme disease
being positive for A. phagocytophilum in a study from Szcze-
cin University (Rymaszewska and Adamska 2011), and 1/79
dogs being positive in a group of apparently healthy sled dogs
(Welc-Falęciak et al. 2009). In addition to the occurrence of
the pathogen in dogs, it is also reported in diverse forms of
wild life in Poland (e.g., roe deer (Welc-Falęciak et al. 2013);
wild boars (Michalik et al. 2012); wild cervids (Hapunik et al.
2011)). Even though this aspect is not examined very often
within the canine population, there is a clear risk of infection
by frequently reported A. phagocytophilum-positive I. ricinus
ticks in Poland.
Autochthonous cases of Ehrlichia canis have so far not
been reported in dogs in Poland, and for Dirofilaria immitis
only one questionable autochthonous case in Poland without
molecular confirmation was described (Światalska and
Demiaszkiewicz 2012).
The vector for E. canis is Rhipicephalus sanguineus
(Groves et al. 1975; Lewis et al. 1977), which in Europe
mainly occurs in places with a Mediterranean climate. For
Poland, only two citations of R. sanguineus occurrence
could be found. One on a mass infestation in an apartment
in Warsaw in the 1970s (Szymański 1979) and one on a
dog in Warsaw, which might be identical with the publica-
tion of the mass infestation in the 1970s, as it is only
mentioned in a review by Nowak-Chmura and Siuda
(2012) without concrete citation. As Poland is not endemic
for R. sanguineus, it can be suggested that E. canis infec-
tion is not autochthonously occurring in Poland, but is
associated with import of dogs or a travel history. Excep-
tionally, imported ticks may establish populations within
all-year temperate homes and subsequently lead to an
“autochthonous”infection, as suggested for Germany
(Dongus et al. 1996).
Apart from the D. immitis case mentioned above,
Dirofilaria repens has so far been detected only in dogs
in central Poland (Demiaszkiewicz et al. 2009) and in dogs
imported from Poland (Pantchev et al. 2011). Typical en-
demic areas for D. immitis are found in the Mediterranean
region extending up to the Alps. As far as the countries
bordering on Poland are concerned, individual cases have
been detected in Slovakia (Iglódyová et al. 2012). Never-
theless, evaluating the temperature records, spanning a 29-
year period (1971–2000), along with the model of Fortin
and Slocombe (1981) modified by Lok and Knight (1998),
for eastern Europe, a threshold value of 130 cumulative
Dirofilaria developing units (DDU) reached in 30 consec-
utive days, being sufficient to facilitate extrinsic incubation
of Dirofilaria, were also recorded for Poland between June
and August and to a very reduced amount as well in
September (Genchi et al. 2011).
The aim of the study described here was to collect current
data on the occurrence and distribution of four major canine
vector-borne pathogens via a large nationwide survey of the
canine population in Poland. A further aim was to characterise
in more detail mixed infections with the various pathogens
and areas of high prevalence.
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Material and methods
General
Serum samples from 3,094 dogs were analysed in the study.
The samples were taken by local veterinarians in 54 partici-
pating veterinary practices distributed throughout all 16 Polish
provinces. The practices were participating in a research pro-
ject which formed part of the “European Project for
Anaplasma and Borrelia Prevalence in Dogs”. The samples
were submitted to a diagnostic laboratory for analysis. The
origin of the dogs was determined using the postcode supplied
with the sample.
Clinical samples, study period, study area
The serum was collected from clinically healthy dogs with a
tick history visiting veterinary practices in all 16 Polish prov-
inces. The samples were collected between March and Octo-
ber 2011.
Laboratory tests, data calculation and visualisation
The samples were picked up from veterinary clinics by PPH
Eskulap, Gliwice, and then submitted to a private veterinary
diagnostic laboratory (IDEXX Vet Med Lab, Ludwigsburg,
Germany) for testing of different CVBDs. Serological testing
was performed using a rapid assay test system (SNAP®
4Dx®, IDEXX Laboratories, Inc., Westbrook, ME, USA)
following the manufacturer’s instructions for use. SNAP®
4Dx® (Fig. 1) is a rapid assay test system based on enzyme
immunoassay technique. The test has been validated for dogs
(Chandrashekar et al. 2010) and is officially registered for use
in dogs in Germany by the Friedrich Loeffler Institute (FLI). A
test unit consists of a coated membrane matrix with five spots
in the reaction area (result window). Three spots are impreg-
nated respectively with a specific peptide antigen of
A. phagocytophilum (a synthetic peptide from the major sur-
face protein (p44/MSP2)), B. burgdorferi s.l. (C6 peptide) and
E. canis (peptides from p30 and p30-1 outer membrane pro-
teins). The D. immitis analyte is derived from antibodies
specific to heartworm antigens, which are primarily produced
by adult females (Weil 1987). The fifth spot serves as a
positive control. A two-chamber system contains wash solu-
tion and substrate solution, which flow across the coated
membrane upon activation of the test (Pantchev et al.
2009a). The sensitivity of the performed test ranges according
to the manufacturer from 99.1 % for A. phagocytophilum and
98.8 % for B. burgdorferi s.l. to 96.2 % for E. canis and
99.2 % for D. immitis, with a specificity for all four pathogens
of 100 % according to Chandrashekar and colleagues (2010).
Antibodies against Anaplasma platys in experimentally infect-
ed dogs have cross-reacted with the A. phagocytophilum an-
alyte, and the E. canis analyte may cross-react with anti-
Ehrlichia chaffeensis antibodies (Chandrashekar et al. 2010).
Nevertheless, one natural A. platys infection in a dog yielded a
negative result in this test (Dyachenko et al. 2012). Cross-
reactivity of the D. immitis analyte in similar commercially
available antigen tests with Angiostrongylus vasorum-positive
dogs has since been described (Schnyder and Deplazes 2012)
and will be discussed later in this publication.
There are a number of different genospecies concerning
B. burgdorferi s.l. There are at least three species in Europe
that are pathogenic for humans: B. burgdorferi sensu stricto,
B. garinii and B. afzelii. The C6 peptide is antigenically
conserved among them and may be used to serodiagnose
borreliosis universally (Liang et al. 2000). Detection of anti-
bodies against C6 peptide in dogs by means of commercially
available tests does not interact with Borrelia vaccination
(O'Connor et al. 2004), succeeds from days 21 to 35 post-
infection onwards (Wagner et al. 2012), and persists in un-
treated dogs for at least 12 months (Levy et al. 2008).
The collected data were analysed by a geographic informa-
tion system (GIS) using the programme RegioGraph 10 (GfK
GeoMarketing, Bruchsal, Germany) to visualise the regional
distribution of collected and analysed serum samples and
antibody- and/or antigen-positive samples for the different
pathogens on administrative maps. Using the three digits as
Fig. 1 Canine SNAP® 4Dx® test
device. Schematic illustration
(left) and photograph of a test
device demonstrated with a
canine serum sample positive for
Borrelia and Anaplasma
antibodies (right)
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points of reference, the locations of positive samples were
displayed on maps with administrative and postcode
boundaries.
The descriptive analysis was performed with the helpof the
validated statistical programme TESTIMATE Version 6.5
from IDV Data Analysis and Study Planning. The presence
of antibodies (for A. phagocytophilum,B. burgdorferi s.l. and
E. canis) or antigen (for D. immitis) for every variable was
dichotomised into negative (=no presence) and positive
(=presence) to calculate the prevalence and the 95 % confi-
dence interval (CI). Additionally, differences from the overall
sum for each of the 16 provinces were calculated using the
Fligner-Wolfe test (many-to-many test, alpha = 0.05) for
A. phagocytophilum,B. burgdorferi s.l. and co-infection with
A. phagocytophilum and B. burgdorferi s.l.
Results
The seropositivity of all tested samples is summarised in
Tab le 1. The overall prevalence of A. phagocytophilum and
B. burgdorferi s.l. in dogs was 12.31 % (n=381; 95 % CI:
11.18–13.52 %) and 3.75 % (n=116; 95 % CI: 3.11–4.48 %),
respectively. The overall prevalence based on the test results
for E. canis and D. immitis in dogs was 0.26 % (n=8;95% CI:
0.11–0.51 %) and 0.16 % (n=5; 95 % CI: 0.05–0.38 %),
respectively.
The results of the D. immitis test component of this study
need to be discussed differentially. Simultaneous use of highly
specific diagnostic methods to differentiate “true”canine
heartworm (D. immitis)and“French”heartworm (A. vasorum,
a potentially fatal canine nematode that also lives as an adult in
the pulmonary arteries) is recommended within overlapping
endemic areas, as some commercially available heartworm
antigen tests show cross-reactivity with A. vasorum
(Schnyder and Deplazes 2012). Nowadays, a revised version
of the test system used in this study, SNAP® 4Dx® Plus
(IDEXX Laboratories, Inc., Westbrook, ME, USA), which
does not show any cross-reactivity between D. immitis and
A. vasorum (Schnyder and Deplazes 2012), and a specific
rapid A. vasorum device (Schnyder et al. 2014) are available,
but they were not on the market when testing was performed
for the present study. Nevertheless, it was possible to follow
the above recommendation of simultaneously using highly
specific diagnostic methods as the dog population in the study
reported here was partially identical with that in a study that
examined dogs for the presence of A. vasorum antibodies and
antigen in Poland (Schnyder et al. 2013). The results of the
two studies were compared.
Looking at the five D. immitis-positive dogs in this study, a
positive A. vasorum antigen sandwich ELISA (Schnyder et al.
2011)andA. vasorum antibody sandwich ELISA (Schucan
et al. 2012) were reported for one dog from Masovia Province,
pointing to potential cross-reactivity between D. immitis and
A. vasorum. No further information for this dog was available,
in particular regarding possible travel to D. immitis-endemic
areas. If this dog needed to be considered in terms of a
potential cross-reaction with A. vasorum, which would be
the case if it had nohistory of travelling abroad, the prevalence
of D. immitis would have to be corrected to 0.13 % (4/3094;
95 % CI: 0.04–0.33 %). The other four dogs (from Warmia-
Masuria, Opole, Greater Poland and Pomerania Provinces)
showed an optical density in the A. vasorum antigen sandwich
ELISA well below the cut-off within the tested population of
Polish dogs (Schnyder et al. 2013) and were thus classified as
A. vasorum-negative in the corresponding study. A negative
A. vasorum antibody ELISA for these dogs in Schnyder and
colleagues (2013)confirmsanA. vasorum-negative status and
thus a true D. immitis-positive result in the performed SNAP®
4Dx® test.
The locations of the positive samples (as coloured spots)
and the sampling area (in dark grey) on the administrative
maps are shown in Figs. 2,3and 4. Furthermore, the number
of positive samples per province is shown in Table 2.
Co-infections with A. phagocytophilum and B. burgdorferi
s.l. were observed in 1.71 % (n=53; 95 % CI: 1.29–2.23 %) of
the tested dogs. One dog proved to have a triple infection with
A. phagocytophilum,B. burgdorferi s.l. and E. canis.The
proportion of single, double and triple infections in the sum
of all positive samples is listed in Table 3.
A travel history or importation cannot be excluded for dogs
positive for E. canis and D. immitis. No co-infections with
D. immitis were recorded.
The epizootiological situation with respect to infections
with A. phagocytophilum and B. burgdorferi s.l. in dogs varies
greatly between individual provinces. The highest percentages
of dogs (more than 20 %) infected with A. phagocytophilum
were noted in Lesser Poland, Silesia and ŁódźProvinces, and
the lowest percentages (below 5 %) in Masovia, Subcarpathia
Tabl e 1 Results of dog serum samples from Poland (n=3,094)tested for
the presence of specific antibodies against Anaplasma phagocytophilum
(Ap), Borrelia burgdorferi s.l. (Bb) and Ehrlichia canis (Ec) and of
circulating antigen of Dirofilaria immitis (Di)
Causative organism Antibody
(Ap, Bb, Ec)
or antigen (Di)
positive dogs/all
tested dogs
Percentage 95 % Confidence
interval
Anaplasma
phagocytophilum
381/3,094 12.31 % 11.18–13.52 %
Borrelia burgdorferi
s.l.
116/3,094 3.75 % 3.11–4.48 %
Ehrlichia canis 8/3,094 0.26 % 0.11–0.51 %
Dirofilaria immitis 5/3,094 0.16 % 0.05–0.38 %
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and Świętokrzyskie Provinces (see also Table 2). Lesser Po-
land, Silesia and ŁódźProvinces have a significantly (in a
descriptive manner) higher prevalence rate than the overall
with respect to A. phagocytophilum, while Masovia,
Subcarpathia, Świętokrzyskie and Lublin Provinces have a
significantly lower prevalence rate than the overall with re-
spect to A. phagocytophilum. The highest prevalence of in-
fection with B. burgdorferi s.l. (>10 %) was noted in dogs
from ŁódźProvince, and the lowest prevalence (<1 %) was
found, just as for A. phagocytophilum, in Masovia Province.
These differences in prevalence from the overall were signif-
icant in terms of a higher prevalence rate than the overall in
ŁódźProvince and a lower prevalence rate than the overall
in Masovia Province for B. burgdorferi s.l. Most cases of
co-infection with A. phagocytophilum and B. burgdorferi
s.l. were observed in ŁódźProvince (8.49 %), while in
Masovia, Subcarpathia and Podlaskie Provinces no such
cases were found (see also Table 2). The higher prevalence
rate than the overall in ŁódźProvince is significant for co-
infection with A. phagocytophilum and B. burgdorferi s.l.
The few dogs positive for E. canis and D. immitis were
distributed throughout the country and no clear regional
focus was found.
Discussion
The main vector of A. phagocytophilum and B. burgdorferi s.l.
in Poland is the castor bean tick, I. ricinus.Itisdistributed
throughout Poland, so that there is a clear potential for trans-
mission of these two pathogens all over the country. Further-
more, both pathogens occur in dogs, can cause clinical disease
in dogs, although for Borrelia so far only pathogenicity of the
genospecies B. burgdorferi s.s. has been proven for dogs
(reviewed by Krupka and Straubinger 2010), and both also
have a zoonotic character. In several studies, mainly concen-
trating on man and the vector tick, the prevalence of both
pathogens has been confirmed in Poland.
Most human cases of borreliosis were originally recorded
in the north-eastern part of the country, but there have since
also been reports of the pathogen from the north-western
region in ticks (Skotarczak et al. 2002,2003; Skotarczak
Fig. 2 Occurrence of Anaplasma phagocytophilum-positive dogs detect-
ed by SNAP® 4Dx® in a population of 3,094 from Poland. Dark grey
areas represent the origin of the tested dog sera. The origins of dogs
positive for circulating A. phagocytophilum antibodies (n= 381) are
shown in red
Parasitol Res
2000; Wodecka and Skotarczak 2000) and of pathogen DNA
also in dogs (Skotarczak and Wodecka 2003,2005). One
study from Poland furthermore confirmed the role of the
genospecies B. burgdorferi s.s. in canine borreliosis
(Wodecka et al. 2009). Vector ticks carrying A. phagocytophilum
have been recorded across the country (e.g., from southern
(Lesser Poland) (Asman et al. 2013) to central (Warsaw)
(Zygner et al. 2008) and northern Poland (Pomerania)
(Stańczak et al. 2004)). Dogs mainly from the north-
westernregionhavebeenreportedtobeA. phagocytophilum-
positive (Skotarczak et al. 2004; Rymaszewska and
Adamska 2011), even though Skotarczak and colleagues
(2004) presumed that the low prevalence data (PCR-based
method) recorded in dogs in that area might indicate that
the domestic dog is not a reservoir for Anaplasma in the
tested region. Nevertheless, in a large German canine
seroprevalence study by Krupka and colleagues (2007),
some of the highest prevalences for A. phagocytophilum in
dogs were detected in the German postcode regions 0 and 1
(23.1%and25.8%),representing the German districts
bordering Poland and the Czech Republic, and thus may
also mirror a focus of A. phagocytophilum in dogs in that
region covering north-west Poland and north-east Germany.
Based on the data for ticks, a more nationwide exposure
seems to occur for A. phagocytophilum,incontrasttoan
apparently more northerly distribution of the borreliosis
pathogen.
The prevalence data from the present study could not
confirm a more northerly occurrence of dogs positive for
B. burgdorferi s.l., but showed by far the highest prevalence
in ŁódźProvince (11.3 %), followed by Opole, Kuyavia-
Pomerania, Silesia and Lower Silesia Provinces, nearly all
located in the south-west of Poland (apart from Kuyavia-
Pomerania). A nationwide occurrence of A. phagocytophilum
in the tested dog population was confirmed, with the lowest
prevalences in the north, north-east, central-east and south-
east (Pomerania, Warmia-Masuria, Podlaskie, Masovia, Lu-
blin, Świętokrzyskie and Subcarpathia Provinces), regions
which in part make up the areas which originally had the
highest occurrence of borreliosis. The highest prevalences
were recorded in the central and southern regions.
Regarding E. canis and D. immitis seropositivity, the few
dogs with a positive reaction (n= 13, or 12 after one potentially
D. immitis/A. vasorum cross-reacting dog had been
Fig. 3 Occurrence of Borrelia burgdorferi s.l. -positive dogs detected by SNAP® 4Dx®in a population of 3,094 from Poland. Dark grey areas represent
the origin of the tested dog sera. The origins of dogs positive for circulating B. burgdorferi s.l. antibodies (n=116) are shown in blue
Parasitol Res
discounted) are distributed throughout the country, so that no
clear focus can be described. The fact that so far E. canis has
not been reported autochthonously in dogs in Poland,
D. immitis has only been published with one questionable
autochthonous case in Poland without molecular confirmation
(Światalska and Demiaszkiewicz 2012), the dogs in the study
that were positive for D. immitis did not show any co-infection
with local agents such as Borrelia/Anaplasma, and finally that
no clear regional focus could be identified, raises the question
of these dogs' travel history and recent import status. Unfor-
tunately no data were available on these aspects.
The cross-reactivity of the D. immitis component of some
commercially available tests in A. vasorum-positive dogs has
been documented (Schnyder and Deplazes 2012). This could
have been a reason for the occurrence of D. immitis-positive
samples in Poland, which has otherwise been negative for
heartworm to date. And indeed, clarification of the results by
using data available from another study, using partially the
same dog population and testing for A. vasorum by using an
antigen and an antibody ELISA (Schnyder et al. 2013),
showed that one out of five dogs reacted positively in the
A. vasorum tests, pointing to a potential cross-reaction. Ex-
amination for microfilariae would have aided discussion ofthe
A. vasorum cross-reaction, as would the travel history of the
dog concerned in areas endemic for D. immitis. But no data on
these aspects were available. Nevertheless, the prevalence of
0.13 % with 4/3,094 dogs reacting positive for D. immitis in
the study presented here is comparable to the prevalence
obtained in an epidemiologically comparable situation in a
large German study, where 4/3,005 dogs were D. immitis-
positive in the SNAP® 4Dx® test (Pantchev et al. (2009b),
based on the data of Krupka et al. (2007)). None of the dogs
positive for E. canis or D. immitis had a co-infection with the
other pathogen, i.e., D. immitis or E. canis.
The number of co-infections with A. phagocytophilum and
B. burgdorferi s.l. has been the subject of several studies in
both man and ticks. As the castor bean tick is the main vector
for both pathogens in Poland, a co-infection could result from
a dually infected tick. This dual infection has been reported for
I. ricinus in northern Poland, e.g., with a prevalence of 8.3 %
of 303 examined adult ticks (Stańczak et al. 2004). However,
it could also be the result of a simultaneous or sequential
Fig. 4 Occurrence of Ehrlichia canis- and Dirofilaria immitis-positive
dogs detected by SNAP® 4Dx® in a population of 3,094 from Poland.
Dark grey areas represent the origin of the tested dog sera. The origins of
dogs positive for circulating E. canis antibodies (n=8) and D. immitis
antigen (n=5) are shown in green and orange,respectively
Parasitol Res
infection by singly infected ticks. In man, the concurrence of
A. phagocytophilum and B. burgdorferi was detected in 3.2 %
in north-eastern Poland (Grzeszczuk et al. 2004) and in 4.5 %
(15/334) (Zwoliński et al. 2004) and 17.5 % (11/63)
(Tomasiewicz et al. 2004), both in mid-eastern Poland. One
of the main concerns in co-infection is the possibility that the
clinical appearance in the patient may be altered, thus poten-
tially making diagnosis more difficult and leading to a more
serious disease outcome (Krupka et al. 2007). Furthermore,
one pathogen might pave the way for another. In one study, for
example, seroreactivity to both A. phagocytophilum and
B. burgdorferi was detected more frequently in suspected
clinical cases in dogs than seroreactivity to either organism
alone (Beall et al. 2008). This again should be borne in mind
by veterinarians across the country, as exposure to both path-
ogens seems possible.
The fact that both are zoonotic pathogens indicates the need
for greater involvement on the part of the public health
authorities.
There are two limitations to this serological survey. First,
the dogs' history of travel abroad and their import status were
not recorded, so that, for the E. canis-andD. immitis-positive
dogs in particular, an autochthonous character cannot be con-
firmed for Poland. Dogs testing positive in a specific area may
have been exposed elsewhere. Second, a positive antibody test
is not necessarily equivalent to the existence of the pathogen
in the canine or vector population of a particular geographic
region; it is only evidence of prior exposure to the correspond-
ing pathogen at some point and some location in the dog’s
history. With respect to the latter limitation, a more differen-
tiated view needs to be taken of the detection of antibodies
against C6 peptide of B. burgdorferi s.l.: The SNAP® 4Dx®
Tabl e 2 Distribution of Anaplasma phagocytophilum (Ap), Borrelia burgdorferi s.l. (Bb), Ehrlichia canis (Ec) and Dirofilaria immitis (Di) positive
samples per province (percentage and total numbers)
Province (number
of veterinary clinics)
Percentage Ap
positive (x/y)
Percentage Bb
positive (x/y)
Percentage Ec
positive (x/y)
Percentage Di
positive (x/y)
Percentage Ap +
Bb positive (x/y)
Percentage Ap +
Bb + Ec positive
(x/y)
Greater Poland (5) 12.72 % (43/338) 2.66 % (9/338) 0.30 % (1/338) 0.30 % (1/338) 0.89 % (3/338)
Kuyavia-Pomerania
(1)
10.26 % (8/78) 6.41 % (5/78) 1.28%c (1/78)
Lesser Poland (4) 25.24 % (53/210) 3.81 % (8/210) 2.86 % (6/210)
Łódź(4) 20.28 % (43/212) 11.32 % (24/212) 8.49 % (18/212)
Lower Silesia (3) 13.39 % (15/112) 5.36 % (6/112) 1.79 % (2/112)
Lublin (5) 7.50 % (24/320) 2.19 % (7/320) 0.94 % (3/320) 0.94 % (3/320)
Lubusz (3) 16.49 % (32/194) 4.64 % (9/194) 0.52 % (1/194) 1.55 % (3/194) 0.52 % (1/194)
Masovia (2) 3.21 % (5/156) 0.64 % (1/156) 0.64 % (1/156) 0.64 % (1/156)/ 0.0 %
(0/156)
a
Opole (1) 15.00 % (18/120) 6.67 % (8/120) 0.83 % (1/120) 1.67 % (2/120)
Podlaskie (2) 9.68 % (12/124) 1.61 % (2/124) 0.81 % (1/124)
Pomerania (6) 8.23 % (20/243) 1.65 % (4/243) 0.41 % (1/243) 0.41 % (1/243) 0.82 % (2/243)
Silesia (3) 20.90 % (42/201) 5.97 % (12/201) 3.48 % (7/201)
Subcarpathia (2) 4.27 % (5/117) 2.56 % (3/117)
Świętokrzyskie (2) 4.80 % (6/125) 1.60 % (2/125) 0.80 % (1/125)
Warmia-Masuria (5) 8.73 % (20/229) 2.62 % (6/229) 0.44 % (1/229) 0.87 % (2/229)
West Pomerania (6) 11.11 % (35/315) 3.17 % (10/315) 0.95 % (3/315)
Total (54) 12.31 % (381/3,094) 3.75 % (116/3,094) 0.26 % (8/3,094) 0.16 % (5/3,094)/0.13 %
(4/3,094)
a
1.71 % (53/3,094) 0.03 % (1/3,094)
xsamples positive for a specific pathogen, ytotal number of samples tested per province
a
Corrected data after discounting one potential A. vasorum cross-reacting dog in Masovia Province
Tabl e 3 Proportion of single, double and triple infections in positive samples (n=456)
Ap (alone) Bb (alone) Ec (alone) Di Ap + Bb Ap + Bb + Ec
Positive samples 328 63 7 5 52 1
Percentage
(95 % CI)
71.93 %
(67.56–76.01 %)
13.82 %
(10.78–17.33 %)
1.54 %
(0.62–3.14 %)
1.10 %
(0.36–2.54 %)
11.40 %
(8.63–14.68 %)
0.22 %
(0.01–1.22 %)
Ap Anaplasma phagocytophilum,BbBorrelia burgdorferi s.l., Di Dirofilaria immitis,EcEhrlichia canis, CI confidence interval
Parasitol Res
test detected infection with Borrelia at the earliest on day 35 or
day 49 post-infection, depending on the dog (Wagner et al.
2012). On the other hand, antibodies to C6 have been detected
in the late stages of infection (>12 months) with a C6 detecting
device (Wagner et al. 2012;Levyetal.2008), and have been
found to decrease significantly after specific treatment, so that
at least for Borrelia the detection of C6 peptide might repre-
sent a more or less robust marker of infection.
Generally, the large number of dogs included and the fact
that two of the pathogens, A. phagocytophilum and
B. burgdorferi s.l., have also been reported in vector ticks,
man or wild life in Poland several times support the conclu-
sion that veterinarians should be aware of infection with these
two pathogens potentially in all Polish provinces. Veterinar-
ians should include these two diseases in their differential
diagnosis and recommend the use of repellents along with
prophylactic measures to prevent disease transmission by
arthropod vectors.
In conclusion, this study represents a nationwide
overview of the occurrence of important canine, but
also zoonotic, pathogens in a large canine population
in Poland. Dogs seropositive for A. phagocytophilum
(12.31 % prevalence) and B. burgdorferi s.l. (3.75 %
prevalence) were detected in all 16 Polish provinces,
even though the prevalence varied between the different
provinces and a slightly more central/southern/mid-west-
ern focus was recorded. Nevertheless, veterinarians
throughout the country should be aware that these two
major canine vector-borne pathogens may occur in their
practice area and exposure of their canine clients is
possible.
E. canis and D. immitis were much less prevalent: 0.26 %
(E. canis) and 0.16 % (D. immitis). As the travel history and
import status of the positive dogs were not available, an
autochthonous character of the latter two pathogens cannot
be confirmed.
Co-infections with A. phagocytophilum and B. burgdorferi
s.l. were recorded in 1.71 % of all examined dogs, and one dog
was even infected with a third pathogen, E. canis.
Acknowledgements The study was funded by Bayer Animal Health
GmbH, Germany. Friederike Krämer is funded in a Bayer project at the
Freie Universität Berlin. The authors are indebted to the veterinarians
participating in this study.
Ethical standards All investigations comply with the current laws of
the countries in which they were performed.
Conflict interests The authors declare that they have no competing
interests.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
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