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.
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ABSTRACT: DNA analysis of blood meals from unfed nymphal Ixodes ricinus allows for the identification of tick host and tick-borne pathogens in the host species. The recognition of host species for tick larvae and the reservoirs of Borrelia, Rickettsia and Anaplasma species were simultaneously carried out by analysis of the blood meals of 880 questing nymphal I. ricinus ticks collected in forest parks of Szczecin city and rural forests in northwestern Poland that are endemic areas for Lyme borreliosis. The results obtained from the study indicate that I. ricinus larvae feed not only on small or medium animals but also on large animals and they (i.e. roe deer, red deer and wild boars) were the most prevalent in all study areas as the essential hosts for larvae of I. ricinus. The composition of medium and small vertebrates (carnivores, rodents, birds and lizards) provided a more diverse picture depending on study site. The reservoir species that contain the most pathogens are the European roe deer Capreolus capreolus, in which two species of Rickettsia and two species of Borrelia were identified, and Sus scrofa, in which one Rickettsia and three Borrelia species were identified. Rickettsia helvetica was the most common pathogen detected, and other included species were the B. burgdorferi s.l. group and B. miyamotoi related to relapsing fever group. Our results confirmed a general association of B. garinii with birds but also suggested that such associations may be less common in the transmission cycle in natural habitats than what was thought previously.Experimental and Applied Acarology 12/2013; · 1.82 Impact Factor
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ABSTRACT: The bacterium Anaplasma phagocytophilum has for decades been known to cause the disease tick-borne fever (TBF) in domestic ruminants in Ixodes ricinus-infested areas in northern Europe. In recent years, the bacterium has been found associated with Ixodes-tick species more or less worldwide on the northern hemisphere. A. phagocytophilum has a broad host range and may cause severe disease in several mammalian species, including humans. However, the clinical symptoms vary from subclinical to fatal conditions, and considerable underreporting of clinical incidents is suspected in both human and veterinary medicine. Several variants of A. phagocytophilum have been genetically characterized. Identification and stratification into phylogenetic subfamilies has been based on cell culturing, experimental infections, PCR, and sequencing techniques. However, few genome sequences have been completed so far, thus observations on biological, ecological, and pathological differences between genotypes of the bacterium, have yet to be elucidated by molecular and experimental infection studies. The natural transmission cycles of various A. phagocytophilum variants, the involvement of their respective hosts and vectors involved, in particular the zoonotic potential, have to be unraveled. A. phagocytophilum is able to persist between seasons of tick activity in several mammalian species and movement of hosts and infected ticks on migrating animals or birds may spread the bacterium. In the present review, we focus on the ecology and epidemiology of A. phagocytophilum, especially the role of wildlife in contribution to the spread and sustainability of the infection in domestic livestock and humans.Frontiers in Cellular and Infection Microbiology 01/2013; 3:31. · 2.62 Impact Factor
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ABSTRACT: We investigated the involvement of birds in the ecology of the western black-legged tick, Ixodes pacificus, and its associated zoonotic bacteria, Borrelia burgdorferi and Anaplasma phagocytophilum, at two interior coast-range study sites in northern California. Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis (GA), and B. burgdorferi s.s., the agent of Lyme disease (LD), are tick-borne pathogens that are well established in California. We screened blood and ticks from 349 individual birds in 48 species collected in 2011 and 2012 using pathogen-specific PCR. A total of 617 immature I. pacificus was collected with almost three times as many larvae than nymphs. There were 7.5 times more I. pacificus at the Napa County site compared to the Yolo County site. Two of 74 (3%) nymphal pools from an Oregon junco (Junco hyemalis) and a hermit thrush (Catharus guttatus) and 4 individual larvae (all from Oregon juncos) were PCR-positive for B. burgdorferi. Blood samples from a golden-crowned sparrow (Zonotrichia atricapilla) and a European starling (Sturnus vulgaris) were positive for A. phagocytophilum DNA at very low levels. Birds that forage on ground or bark and nest on the ground, as well as some migratory species, are at an increased risk for acquiring I. pacificus. Our findings show that birds contribute to the ecologies of LD and GA in California by serving as a blood-meal source, feeding and transporting immature I. pacificus, and sometimes as a source of Borrelia infection.Ticks and Tick-borne Diseases 06/2014; · 2.35 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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