PCR detection of granulocytic Anaplasma and Babesia in Ixodes ricinus ticks and birds in west-central Poland.
ABSTRACT The aim of the study was to establish the role of forest birds as reservoirs of Anaplasma phagocytophilum and Babesia spp. in Wielkopolski National Park. A total of 108 birds from 9 species were collected between May-September 2002. Blood samples were taken from 84 specimens and 442 individuals of the common tick, Ixodes ricinus, were collected from the birds. The 73 additional ticks were collected from vegetation. PCR amplification of a fragment of the epank 1 gene and 18S rRNA gene was used for detection of A. phagocytophilum and Babesia spp. DNA, respectively. Pathogen DNA was not detected in any of the blood samples or ticks collected from birds. On the other hand, 3 ticks collected from vegetation (4.1% of all examined specimens) were positive for A. phagocytophilum DNA. In spite of the high level of infestation of birds by I. ricinus, it is clear that they do not constitute a competent reservoir of A. phagocytophilum and Babesia in WNP. Additionally, I. ricinus is not a significant vector in this area.
- SourceAvailable from: Pilar Alberdi[show abstract] [hide abstract]
ABSTRACT: Samples of blood, spleen and legs from 112 culled roe deer (Capreolus capreolus) were collected from nine sites widespread in the United Kingdom. The prevalence of infection with Ehrlichia phagocytophila was determined by serology and polymerase chain reaction. Means of 58% of 102 plasma or serum samples were seroreactive by IFA, 38% of 84 blood samples and 29% of 82 spleen samples were positive by PCR. Ticks on legs of 71 roe deer were Ixodes ricinus larvae, nymphs and adults and 83% of legs were infested. Numbers of ticks corresponded positively to the percentage of samples positive for E. phagocytophila by serology and PCR for different sampling sites. Ixodes ricinus nymphs collected from the vegetation at one site with infected deer were analysed for infection with E. phagocytophila by examination of Feulgen stained salivary glands. Of 135 nymphs 5% were infected. These results confirm that roe deer are commonly parasitized by both E. phagocytophila and its vector tick in such a way that it is likely to be an important natural mammalian reservoir of E. phagocytophila.Epidemiology and Infection 05/2000; 124(2):315-23. · 2.87 Impact Factor
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ABSTRACT: The presence and distribution of Ehrlichia spp. and Borrelia burgdorferi sensu lato was demonstrated among ixodid ticks collected in the Baltic regions of Russia, where Lyme borreliosis is endemic. A total of 3,426 Ixodes ricinus and 1,267 Ixodes persulcatus specimens were collected, and dark-field microscopy showed that 265 (11.5%) I. ricinus and 333 (26.3%) I. persulcatus ticks were positive. From these samples, 472 dark-field-positive and 159 dark-field-negative ticks were subjected to PCR and subsequent reverse line blot hybridization. Fifty-four ticks (8.6%) carried Ehrlichia species, and 4 (0.6%) carried ehrlichiae belonging to the Ehrlichia phagocytophila complex, which includes the human granulocytic ehrlichiosis agent. The E. phagocytophila complex and an Ehrlichia-like species were detected only in I. ricinus whereas Ehrlichia muris was found exclusively in I. persulcatus, indicating a possible vector-specific infection. Borrelia garinii was found predominantly in I. persulcatus, but Borrelia afzelii was evenly distributed among the two tick species. Only two I. ricinus ticks carried B. burgdorferi sensu stricto, while Borrelia valaisiana and a newly identified B. afzelii-like species were found in 1.7 and 2.5% of all ticks, respectively. Of the dark-field-positive ticks, only 64.8% yielded a Borrelia PCR product, indicating that dark-field microscopy may detect organisms other than B. burgdorferi sensu lato. These observations show that the agent of human granulocytic ehrlichiosis may be present in ticks in the Baltic regions of Russia and that clinicians should be aware of this agent as a cause of febrile disease.Journal of Clinical Microbiology 07/2001; 39(6):2237-42. · 4.07 Impact Factor
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ABSTRACT: During the spring of 1996, an estimated 581,395 Ehrlichia-infected ticks were imported into Sweden by migrating birds. Ehrlichia gene sequences found in ticks collected from these migrating birds were identical to those of granulocytic ehrlichiosis found in domestic animals and humans in Sweden. These findings support the idea that birds may play a role in dispersing Ehrlichia.Emerging infectious diseases 7(5):877-9. · 5.99 Impact Factor
ORIGINAL ARTICLES AAEM
Ann Agric Environ Med 2006, 13, 21–23
Received: 1 November 2004
Accepted: 30 May 2005
PCR DETECTION OF GRANULOCYTIC ANAPLASMA AND BABESIA
IN IXODES RICINUS TICKS AND BIRDS IN WEST-CENTRAL POLAND
?????????? Adamska, Agnieszka Maciejewska
Department of Genetics, Szczecin University, Szczecin, Poland
Skotarczak B, Rymaszewska A, Wodecka B, Sawczuk M, Adamska M, Maciejewska
A: PCR detection of granulocytic Anaplasma and Babesia in Ixodes ricinus ticks and
birds in west-central Poland. Ann Agric Environ Med 2006, 13, 21–23.
Abstract: The aim of the study was to establish the role of forest birds as reservoirs of
Anaplasma phagocytophilum and Babesia spp. in Wielkopolski National Park. A total
of 108 birds from 9 species were collected between May–September 2002. Blood
samples were taken from 84 specimens and 442 individuals of the common tick, Ixodes
ricinus, were collected from the birds. The 73 additional ticks were collected from
vegetation. PCR amplification of a fragment of the epank 1 gene and 18S rRNA gene
was used for detection of A. phagocytophilum and Babesia spp. DNA, respectively.
Pathogen DNA was not detected in any of the blood samples or ticks collected from
birds. On the other hand, 3 ticks collected from vegetation (4.1% of all examined
specimens) were positive for A. phagocytophilum DNA. In spite of the high level of
infestation of birds by I. ricinus, it is clear that they do not constitute a competent
reservoir of A. phagocytophilum and Babesia in WNP. Additionally, I. ricinus is not a
significant vector in this area.
Address for correspondence: ?????? ???????? Skotarczak, Szczecin University,
Faculty of Biology, Departament of Genetics, Al. Piastów 40B, 71-065 Szczecin,
Poland. E-mail: Bogumila_Skotarczak@sus.univ.szczecin.pl
Key words: Anaplasma and Babesia in birds, Anaplasma and Babesia in ticks, PCR.
A new systematic classification of ehrlichia has been
implemented recently, based on genetic analysis of 16S
rRNA, groESL and surface proteins. The former Ehrlichia
phagocytophila, Ehrlichia equi and the human granulo-
cytic ehrlichiosis agent are currently assigned to the same
species Anaplasma phagocytophilum because of their
genetic similarity . This pathogen is widely distributed
in Europe and North America. Its reservoirs are forest
dwelling rodents and ruminants [1, 4]; birds have also
been considered as potentially maintaining A. phagocyto-
philum in the environment [2, 3]. Numerous studies have
shown that the common tick, Ixodes ricinus, is the main
vector of anaplasma in Europe [5, 6, 10, 19]. This tick
also transmits other pathogens such as Babesia [11, 15, 16].
Our previous studies in a forest habitat of the Wielko-
polski National Park (WNP) have shown that yellow-nec-
ked mice (Apodemus flavicollis) are a competent reservoir
of Borrelia burgdorferi sensu lato, and also that feeding
I. ricinus ticks are competent vectors for B. burgdorferi
s.l. and A. phagocytophilum [12, 17]. In the present study,
the role of forest birds as reservoirs of A. phagocytophi-
lum and Babesia spp. is explored in WNP.
MATERIAL AND METHODS
Birds were caught in ornithological nets (permit no.
DLOPiKog.4201/154/00). The nets were checked at least
every 2 hours from sunrise to sunset during four
consecutive days between May–September 2002. The
captured specimens were put in cloth bags and transported
Skotarczak B, Rymaszewska A, Wodecka B, Sawczuk M, Adamska M, Maciejewska A
to a field station. Ticks were removed from infested birds
and blood (about 2 µl) was drawn by hypodermic needle
and pipette from the shoulder vein. Birds were kept alive
through the entire process. A total of 108 birds from 9
species were captured. Blood samples were drawn from
84 of these. 442 specimens of I. ricinus ticks (291
nymphs and 151 larvae) were collected from the birds,
and an additional 73 ticks (33 imago and 8 pools consist
of 5 nymphs) were collected from vegetation. The DNA
was extracted from ticks with the method described by
Guy and Stanek (1991). The DNA from bird blood was
isolated using the Master PureTM DNA Purification Kit
(Epicentre, USA) and stored at –70ºC.
Detection of A. phagocytophilum DNA. For the epank
1 gene, a fragment of 444 bp was amplified by using
primers LA1 and LA6 .
Detection of Babesia DNA. A fragment of the gene
encoding the nuclear small-subunit ribosomal RNA (SS-
rDNA) was used as a target with primers 1FOR(5'-
TGTCTTAAAGATTAAGCCATGCATGT-3') and 1REV
(5'-TTGTGA ACC TTATCACTTAAAGGAAG-3') with
an expected product size of 1650 bp. These primers were
constructed by our group. The time/temperature profiles
of the PCR’s were the same as described earlier .
PCR products were separated by electrophoresis in 2%
agarose (ICN, USA) and stained with ethidium bromide.
For molecular size assessment, a mass marker was used
The 108 captured birds were assigned to the following
species (number of specimens in parentheses): Cocco-
thraustes coccothraustes (6), Erithacus rubecula (6),
Fringilla coelebs (10), Parus caeruleus (1), Parus major
(11), Sitta europaea (6), Sturnus vulgaris (20), Turdus
philomelos (24), T. merula (24). The largest number of
ticks was collected from T. philomelos – 86 nymphs and
51 larvae, and also T. merula – 80 nymphs and 35 larvae.
25 larvae were collected from F. coelebs (chaffinch). The
smallest number of ticks was collected from P. caeruleus
(blue tit) - 2 and S. europaea (nuthatch) – 8. (Fig. 1). No
larvae were present on the blue tit and nuthatch. A total of
73 ticks I. ricinus were taken from vegetation, the largest
number in June (41.1%), the smallest in September
(9.6%). During July, no ticks were collected from the
vegetation, which may have been the result of high air
temperature and low humidity. From 73 individuals of
I. ricinus were fixed in 41 isolates (33 imago and 8 pools
consist of 5 nymphs). Pathogen DNA was not detected in
any of the blood samples or ticks collected from birds. On
the other hand, 3 females of ticks collected from
vegetation (4.1% of all examined specimens, 9.1% of
imago) were positive for A. phagocytophilum DNA.
In Poland, only a few areas have been screened for the
level of infection of I. ricinus by A. phagocytophilum.
Studies from the northwest and northeast have shown that
the mean level of infection varies between 1.4% in the
Zachodniopomorskie Province ??????????????????????????
forest  and 19.2% in Pomorskie Province . Areas
inhabited by ticks also support many species of animals
potentially serving as reservoirs for A. phagocytophilum
or the Babesia species. The zoonotic reservoir of
A. phagocytophilum is still being explored; it is assumed
that small mammals, birds and game animals are
reasonable candidates . This study, screening blood
collected from birds for A. phagocytophilum, has shown
that birds are not reservoirs of this pathogen in WNP.
A previous study has also ruled out rodents . Game
animals are probably the most important in this respect.
Bird reservoirs of A. phagocytophilum have so far been
little studied. Alekseev et al.  studied infection in ticks
collected from 8 species of migrating passerine birds.
These birds were captured in the Kaliningrad district
during spring and autumn 2000. A total of 1606 birds
were captured but ticks were removed from only 110
(6.8%). Pathogen DNA was detected in 14% of the
examined ticks. Alekseev et al.  suggest that the human
granulocytic agent was acquired by co-feeding, not from
an infected bird.
Bjöersdorff et al.  conducted a similar study in
Sweden. From 3054 passerine birds, only 73 (from 18
species) were infested by ticks, from which 165 individu-
als of I. ricinus were collected. Anaplasma DNA was
detected in 8% of nymphs. A study establishing the
significance of pheasant, Phasianus colchicus, as a zoo-
notic reservoir was conducted in England [8, 13]. Phea-
sants were host to Borrelia but were not a competent
reservoir of Anaplasma because this pathogen was not
present in any of the studied birds.
From literature published to date, it seems that birds
from several families can be hosts of many species of
Babesia. According to a review by Peirce , the most
common pathogens of birds are: B. ardeae, B. avium,
Number of I. ricinus specimens
Birds species: C.c. - Coccothraustes coccothraustes, E.r. - Erithacus
rubecula, F.c. - Fringilla coelebs, P.c. - Parus caeruleus, P.m. - Parus
major, S.e. - Sitta europaea, S.v. - Sturnus vulgaris, T.p. - Turdus
philomelos, T.m. - Turdus merula
Figure 1. Number of ticks I. ricinus collected from birds from Wielko-
polski National Park (May–September 2002).
Anaplasma and Babesia in Ixodes ricinus ticks and birds
B. balearicae, B. bennetti, B. emberizica, B. frugilegica,
B. henryi, B. kazachstanica, B. krylovi, B. moshkovskii,
B. mujunkumica, B. peircei, B. polea, B. rustica, B. shortti
and B. socius. The PCR primers used in the present study
allow for the detection of most of these pathogens,
however, a negative result was obtained in all samples.
This may be caused by the small number of birds
collected from each species. These pathogens seem to be
highly species-specific in their choice of host.
We thank dr Jerzy Michalik from Adam Mickiewicz
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