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Veterinary Parasitology 153 (2008) 139142

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Short communication
Prevalence of Babesia canis, Borrelia afzelii, and Anaplasma
phagocytophilum infection in hard ticks removed from dogs
in Warsaw (central Poland)
Wojciech Zygner a,*, Sawomir Jaros b, Halina Wedrychowicz a,b
a
Division of Parasitology and Parasitic Diseases, Department of Preclinical Sciences,
Faculty of Veterinary Medicine, Warsaw Agricultural University, Ciszewskiego 8, 02-786 Warsaw, Poland
b
W. Stefanski Institute of Parasitology, Twarda 51/55, 00-818 Warsaw, Poland
Received 18 October 2007; received in revised form 16 January 2008; accepted 23 January 2008

Abstract
The purposes of this study were to specify the occurrence and prevalence of Babesia canis, Borrelia burgdorferi sensu lato, and
Anaplasma phagocytophilum in ticks removed from dogs in Warsaw, and to determine the Borrelia species occurring in Ixodes
ricinus ticks. Among 590 collected ticks, 209 were identified as I. ricinus, and 381 as Dermacentor reticulatus. DNA of B. canis was
detected in 11% of D. reticulatus ticks. We found that 6.2% of I. ricinus ticks harbored B. burgdorferi s.l. specific DNA and 2.9%
harbored A. phagocytophilum DNA. In these samples sequencing of the detected Borrelia amplicon confirmed infection with
Borrelia afzelii genospecies. New sequences were submitted to the GenBank1 database (accession no. EU152128, EU152127,
EU152126). This work is the first detection of B. afzelii and A. phagocytophilum in ticks from Warsaw, and the first survey for the
prevalence of B. canis, B. afzelii, and A. phagocytophilum in ticks in central Poland.
# 2008 Elsevier B.V. All rights reserved.

Keywords: Anaplasma phagocytophilum; Babesia canis; Borrelia afzelii; Ticks; Poland; Warsaw

1. Introduction Ixodes ricinus ticks on dogs (Zygner and Wedrycho-

Hard ticks are ectoparasites of humans and animals,
which are distributed worldwide. They are vectors of
many viral, rickettsial, bacterial and protozoan patho-
gens. Depending on the geographical region the
invasion of ticks in dogs implicates the risk of various
canine tick-borne diseases. A previous study in Warsaw
revealed the occurrence of Dermacentor reticulatus and
The work presented here is a part of Wojciech Zygners doctoral
studies at Warsaw Agricultural University.
* Corresponding author. Tel.: +48 22 593 60 45;
fax: +48 22 593 60 48.
E-mail address: wojciechzygner@yahoo.pl (W. Zygner).
wicz, 2006). This study as Foldvari et al. (2007) and
Sreter et al. (2005) showed geographical expansion of
D. reticulatus, which transmits Babesia canis. Other
species of genus Babesia infecting dogs are transmitted
by ticks from genus Rhipicephalus and Haemaphysalis
which do not occur in Poland (Uilenberg, 2006). I.
ricinus is the most prevalent tick species in Poland,
which can transmit Borrelia burgdorferi sensu lato and
Anaplasma phagocytophilum (Grzeszczuk and Stanc-
zak, 2006).
Canine babesiosis in Europe is caused by B. canis, B.
vogeli, and B. gibsoni (Uilenberg, 2006), but in Poland
only B. canis has been detected in dogs (Sobczyk et al.,
2005). In this study the prevalence of B. canis infection
in dogs was not estimated. In Europe Lyme borreliosis

0304-4017/$ see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2008.01.036
140 W. Zygner et al. / Veterinary Parasitology 153 (2008) 139142
in dogs can be caused by the infection with Borrelia
genospecies such as B. burgdorferii sensu stricto, B.
garinii and B. afzelii (Hovius, 2005). Cases of
autochthonous B. burgdorferii sensu stricto and A.
phagocytophilum infections of dogs have been reported
in Poland, but these are single studies from northern
Poland (Skotarczak et al., 2004; Skotarczak and
Wodecka, 2005). There is no study in Poland in which
the prevalence of these pathogens in ticks removed from
dogs have been estimated.
The aim of this study is to continue the previous
survey (Zygner and Wedrychowicz, 2006), in which we
detected two species of ticks in dogs from Warsaw.
Among all collected ticks 35.4% were identified as I.
ricinus and 64.6% as D. reticulatus. In the presented
study the authors estimated the prevalence of B. canis
infection in D. reticulatus ticks and the prevalence of B.
burgdorferi sensu lato and A. phagocytophilum in I.
ricinus collected in the previous survey.
2. Materials and methods
During 2 years, from March 2003 to February 2005,
ticks were collected in Warsaw veterinary clinics from
dogs presented for veterinary care. Among 590
collected ticks, 381 were identified as D. reticulatus,
and among them, 137 as male and 244 as female
specimens. The other 209 out of 590 ticks were
identified as I. ricinus, and among them, 16 specimens
were males and 193 were females. Larval or nymphal
stages of ticks were not found in these dogs (Zygner and
Wedrychowicz, 2006). All collected ticks were kept at
70 8C (Jouan1 VX 530 Series 2) until the isolation of
DNA was performed. Before DNA extraction ticks were
washed in 70% ethanol and sterile water, they were then
homogenized in 100 ml PBS with sterile pestle. DNA
was extracted from individual ticks using the Genomic
Mini kit (A&A Biotechnology) according to the
manufacturers instructions, preceded by 6 h digestion
in Proteinase K. The efficiency of DNA isolation was
confirmed by electrophoresis in a 1.5% agarose gel.
Isolated DNA was stored at 70 8C.
Lysates from D. reticulatus were used to detect DNA
of B. canis. PCR was performed according to Sobczyk
et al. (2005) with the primers BcW-A (50 -CAT CTA AGG
AAG GCA GCA GG-30 ) and BcW-B (50 -TTA ATG GAA
ACG TCC TTG GC-30 ) used to amplify the 18S rDNA
gene fragment of B. canis. The expected product was
about 500 bp in size. As a positive control we used DNA
lysate from the blood of dogs infected with B. canis. The
infection was confirmed by sequencing of the PCR
product, which revealed to be 100% identical with a
fragment of the B. canis 18S ribosomal DNA gene under
accession no. AY321119 in the GenBank1 database.
Lysates from I. ricinus were used to detect DNA of B.
burgdorferii s.l. and A. phagocytophilum. PCR was
performed according to Skotarczak et al. (2005) and
Walls et al. (2000) with the primers: SC1 (50 -GCT GTC
AGT GCG TCT TAA-30 ) and SC2 (50 -CTT AGC TGC
TGC CTC CGT A-30 ) used to amplify the 16S rDNA
gene fragment of B. burgdorferi s.l., and LA6 (50 -
GAGAGA TGC TTA TGG TAA GAC-30 ) and LA1 (50 -
CGT TCA GCC ATC ATT GTG AC-30 ) used to amplify
the epank1 gene fragment of A. phagocytophilum. The
expected products were about 300 bp in size for B.
burgdorferii s.l. and about 450 bp for A. phagocyto-
philum. As a positive control we use DNA lysates from
roe deer (Capreolus capreolus) infected with B.
burgdorferii sensu stricto and from I. ricinus tick
infected with A. phagocytophilum obtained from
University of Szczecin, Poland. All PCR was carried
out in MJ Research PTC200 thermal cycler.
The size of the PCR product was analyzed by
electrophoresis in a 1.5% agarose gel stained with
ethidium bromide. The PCR products with the expected
amplicon size were isolated from the agarose gel using
the Gel-Out kit (A&A Biotechnology). Next, all PCR
products were sequenced to verify the presence of B.
burgdorferii s.l. and A. phagocytophilum DNA. Nine
out of 42 B. canis PCR products were randomly selected
for sequencing (three products obtained from male and
six obtained from female D. reticulatus ticks). The
sequencing reaction was carried out on the AbiPrism1
Genetic Analyser using computer programme GeneS-
can1 Analysis Software. The obtained sequences were
compared to sequence data available in the GenBank1
using the BLASTN 2.2.17 program (http://
www.ncbi.nlm.nih.gov/BLAST/). New sequences were
submitted to the GenBank1 database.
3. Results
Babesia DNA was amplified from 42 out of 381
(11%) samples of D. rerticulatus ticks. The nine
selected product sequences showed 99% similarity with
18S rDNA partial sequence of B. canis canis isolated
from dogs in Poland (accession no. AY321119).
Borrelia DNA was detected in 13 out of 209 (6.2%)
samples of I. ricinus ticks. These 13 products were
sequenced. The sequences showed 99% similarity with
16S rDNA partial sequence of B. afzelii (accession no.
DQ111061).
Anaplasma DNA was detected in 6 out of 209 (2.9%)
samples of I. ricinus ticks. These six products were
 W. Zygner et al. / Veterinary Parasitology 153 (2008) 139142
Table 1
Sequences of Babesia canis, Borrelia afzelii, and Anaplasma phagocytophilum submitted to the GenBank1 database
Pathogen Accession no. Sequence
length (bp)
B. canis EU152128 509
B. afzelii EU152127 243
A. phagocytophilum EU152126 398
Table 2
The number of ticks which harbored Babesia canis, Borrelia afzelii, and Anaplasma phagocytophilum DNA (number of ticks which harbored the
DNA of a pathogen/total number of ticks (%); -, tick species which do not transmit a pathogen)
Pathogen Dermacentor reticulatus
Male
B. canis 13/137 (9.5)
B. afzelii
A. phagocytophilum
sequenced. The sequences showed 99% similarity with
ankryin partial gene of A. phagocytophilum (accession
no. AY529488).
Coinfections of B. afzelii and A. phagocytophilum
were not found in I. ricinus ticks in this research. New
sequences submitted to GenBank1 are presented in
Table 1. The number of ticks, both male and female, and
specimens which harbored DNA of each pathogen
species are presented in Table 2.
4. Discussion
The purposes of this study were to specify the
occurrence and prevalence of B. canis, B. burgdorferi
sensu lato, and A. phagocytophilum in ticks removed
from dogs in Warsaw, as well as to determine the
Borrelia species occurring in I. ricinus ticks.
The occurrence of canine babesiosis in Warsaw was
confirmed in a previous study (Sobczyk et al., 2005). In
the presented study we detected B. canis in D.
reticulatus ticks the first time in Poland. This work is
also the first molecular detection of B. canis infection in
male D. reticulatus ticks. In previous studies Foldvari
et al. (2007) investigated the occurrence of Babesia
DNA in female D. reticulatus ticks in Hungary, and Duh
et al. (2006) did not find Babesia species in male D.
reticulatus ticks in Slovakia. In the presented study
9.5% of male D. reticulatus ticks harbored B. canis
DNA. The results of this study differ from those from
Hungary and Slovakia where Foldvari et al. (2007)
found in total 29.9% positive ticks and Duh et al. (2006)
detected B. canis DNA only in 1% of ticks. The high
prevalence of D. reticulatus ticks in Warsaw (Zygner
141
Similarity to sequences Accesion no. of sequences
present previously in present previously in the
the GenBank1 database (%) GenBank1 database
99 AY321119
99 DQ111061
99 AY529488
Ixodes ricinus
Female Male Female
29/244 (11.9)
2/16 (12.5) 11/193 (5.7)
1/16 (6.2) 5/193 (2.6)
and Wedrychowicz, 2006) and the high prevalence of B.
canis infection in this tick species can cause a
considerable number of infections with B. canis in
dogs in Warsaw. To the authors knowledge there is no
research on prevalence of canine babesiosis in Poland.
Furthermore, studies are necessary to determine the
prevalence of B. canis infection in dogs.
Comparing the infection rate of B. canis, the
prevalence of B. afzelii in the collected I. ricinus ticks
was rather low. The prevalence of spirochetes in the
present work is in agreement with the results obtained by
Stanczak et al. (1999) in north-eastern Poland and
Foldvari et al. (2007) in Hungary. In those studies 5% of I.
ricinus ticks harbored B. burgdorferi senu lato DNA in
Poland and 5.5% of I. ricinus ticks harbored B. afzelii or B.
garinii DNA in Hungary. This work is the first molecular
detection of B. afzelii in ticks from Warsaw. The
occurrence of this pathogen threatens humans and
animals. To the authors knowledge there is no study
on the prevalence and the occurrence of human or canine
borreliosis in Warsaw, and further studies are necessary to
estimate the risk of the disease both in dogs and in humans.
The prevalence of A. phagocytophilum in I. ricinus
ticks was the lowest among detected pathogens. This
result differs from the results obtained by Stanczak et al.
(2004) and Grzeszczuk and Stanczak (2006) in northern
and north-eastern Poland where 1423.7% I. ricinus ticks
harbored A. phagocytophilum DNA. The difference can
result from the fact that in those studies ticks were
collected in woodlands and suburban areas where the
reservoir of the pathogen exists. The present work is the
first detection of A. phagocytophilum in Warsaw, which
like B. afzelii is a threat to infection of humans and
142 W. Zygner et al. / Veterinary Parasitology 153 (2008) 139142
animals. The prevalence of anaplasmosis, like borrelio-
sis, has never been estimated in humans and animals in
Warsaw, and there is need for further research in this area.
There is a possibility that the collected ticks may have
become infected with these three pathogens during
feeding on dogs. However, it is also possible that I.
ricinus ticks may have become infected with B. afzelii or
A. phagocytophilum while they were feeding on rodents
or other hosts in earlier stages of their development. In
addition D. reticulatus ticks may have become infected
with B. canis as a result of transovarial transmission. To
sum up, ticks infesting dogs in Warsaw are vectors of B.
canis, B. afzelii, and A. phagocytophilum.
5. Conclusions
This is the first study in Warsaw in which the
prevalence of B. canis, B. afzelii, and A. phagocyto-
philum was evaluated and the occurrence of B. afzelii
and A. phagocytophilum was confirmed. This work is
also the first PCR survey of B. canis in D. reticulatus
ticks in Poland and the first detection of this pathogens
DNA in D. reticulatus male ticks in the world. The
occurrence of pathogens, detected in this work, threaten
both humans and animals in Warsaw.
Acknowledgements
The authors would like to acknowledge the positive
control samples of B. burgdorferii sensu stricto and A.
phagocytophilum provided by Dr. Beata Wodecka
(Department of Genetics, Faculty of Biology, Szczecin
University, Poland). We would also like to thank to
veterinarian from Warsaw for their assistance in
collection of ticks from dogs.
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