Anaplasma phagocytophilum in Ixodes ricinus ticks and human granulocytic anaplasmosis seroprevalence among forestry rangers in Białystok region.

Abstract

Human granulocytic anaplasmosis, former ehrlichiosis, is a tick-borne zoonosis of increasing recognition. The aim of the study was: 1) to assess the prevalence of Anaplasma phagocytophilum infection in Ixodes ricinus ticks collected in recreational forests in Bialystok vicinity, the capital of podlaskie voivodship; 2) to evaluate the prevalence of IgG and IgM antibodies to A. phagocytophilum among forestry rangers from the same region.
Of the 372 ticks examined, 54 (14.5%) yield the positive PCR reaction. The highest prevalence was detected in females, up to 27.8% (37/133), almost one third lower in males--9.2% (13/142), followed by nymphs--4.1% (4/97). Human seropositivity study revealed IgG antibodies against A. phagocytophilum in 9 out of 231 individuals (3.9%). No IgM antibodies were found. Sixty-seven individuals 67/231 (29%) reported erythema migrans. IgM anti-Borrelia burgdorferi antibodies were detected in 32 out of 121 (26.4%) persons tested, IgG--in 43 out of 231 (18.6%).
The data obtained show relatively low A. phagocytophilum seroreactivity among professionally exposed to tick group of forestry workers despite high A. phagocytophilum infection level in the competent vector--I. ricinus ticks.

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Anaplasma phagocytophilum in Ixodes ricinus ticks and human granulocytic anaplasmosis seroprevalence among forestry rangers in Białystok region
· Advances in Medical Sciences · Vol. 51 · 2006 ·
* CORRESPONDING AUTHOR:
Department of Infectious Diseases
Medical University of Białystok
ul. urawia 14, 15-540 Białystok, Poland
Tel: +48 85 7409479, Fax: +48 85 7416421
e-mail: oliwa@amb.edu.pl (Anna Grzeszczuk)
Received 14.06.2006 Accepted 21.07.2006
Anaplasma phagocytophilum in Ixodes ricinus ticks
and human granulocytic anaplasmosis seroprevalence among
forestry rangers in Białystok region
Grzeszczuk A
Department of Infectious Diseases, Medical University of Białystok, Poland
Abstract
Purpose: Human granulocytic anaplasmosis, former ehrli-
chiosis, is a tick-borne zoonosis of increasing recognition.
The aim of the study was: 1) to assess the prevalence of
Anaplasma phagocytophilum infection in Ixodes ricinus ticks col-
lected in recreational forests in Białystok vicinity, the capital of
podlaskie voivodship; 2) to evaluate the prevalence of IgG and
IgM antibodies to A. phagocytophilum among forestry rangers
from the same region.
Results: Of the 372 ticks examined, 54 (14.5%) yield the
positive PCR reaction. The highest prevalence was detected in
females, up to 27.8% (37/133), almost one third lower in males
– 9.2% (13/142), followed by nymphs – 4.1% (4/97). Human
seropositivity study revealed IgG antibodies against A. phago-
cytophilum in 9 out of 231 individuals (3.9%). No IgM antibod-
ies were found. Sixty-seven individuals 67/231 (29%) reported
erythema migrans. IgM anti-Borrelia burgdorferi antibodies were
detected in 32 out of 121 (26.4%) persons tested, IgG – in 43 out
of 231 (18.6%).
Conclusions: The data obtained show relatively low A. pha-
gocytophilum seroreactivity among professionally exposed to
tick group of forestry workers despite high A. phagocytophilum
infection level in the competent vector – I. ricinus ticks.
Key words: Anaplasma phagocytophilum, Ixodes ricinus, ticks,
antibodies, human granulocytic anaplasmosis, forestry rangers,
Białystok region, Poland.
Introduction
Anaplasma phagocytophilum is a unique bacterium infect-
ing and multiplying successfully in granulocytes of broad range
of hosts, including domestic and wild animals – canids, horses,
sheep, cattle, European bisons and rodents, as well as humans.
Infection may be subclinical or manifesting as a non specific
febrile disease, called granulocytic anaplasmosis, tick fever or
pasture fever in sheep [1-3]. A. phagocytophilum is transmitted
by ticks from Ixodes persulcatus complex, which in Europe is
mainly I. ricinus [1,4]. Ticks and tick-borne diseases are endemic
in north-eastern Poland. This region has also the highest
incidence of tick-borne encephalitis in Poland – 7.8/100 000 vs
0.46/100 000 for the whole country in 2005 and Lyme borreliosis
(63.1 vs 11.5) [5]. Ecological and socio-economical changes in
our region lead to increased ticks abundance and therefore
augmented exposure of human population to tick transmitted
pathogens.
Forestry workers constitute the professional group greatly
exposed to ticks. The aim of the study was: 1) to assess the
prevalence of A. phagocytophilum infection in I. ricinus ticks
collected in recreational forests in Białystok vicinity, the capital
of podlaskie voivodship; 2) to evaluate the prevalence of IgG
and IgM antibodies to A. phagocytophilum among forestry rang-
ers from the same region.
Material and methods
Ticks
Host seeking ticks were collected by flagging lower vegeta-
tion in different forested areas in Białystok vicinity – Pietrasze,
Strzelnica, Dzikie and in Knyszyn Primeval Forest – Bobrowa,
Korytne, Supraśl-Pólko, Królowy Most and in Biebrza National
Reserve, Tab. 1. Collected ticks were individually evaluated
prior to DNA extraction by a qualified entomologist with regard
to species and gender according to Siuda [6]. The ticks were
killed in hot water, placed in separate vials (adult) or pooled by

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Grzeszczuk A
2-5 (nymphs) and fixed in 70% ethanol for further investigation
by PCR for the presence of A. phagocytophilum.
Human study population
Study participants were recruited from the following forest
inspectorates localized in Białystok vicinity: Dojlidy, Supraśl,
Czarna Białostocka, Waliły in July, August 2004 or 2005. Serum
samples were collected from 231 forestry workers, 40 females
and 191 males, aged 49±12 years. The questionnaire regarding
age, length and character of employment in the forest, Lyme
borreliosis and tick-borne encephalitis history, ticks exposure
and actual complains such as fever, arthralgia, mialgia was filled
by a physician.
DNA extraction
DNA was extracted by the ammonium hydroxide lysis
(NH4OH) according to Rijpkema et al. [7]. Lysates were stored
in -20°C until examination.
Polymerase Chain Reaction
PCR was performed according to Pancholi et al. [8]. The
primers EHR 521 (5’-TGT AGG CGG TTC GGT AAG TTA
AAG-3’) and EHR 747 (5’-GCA CTC ATC GTT TAC AGC
GTG-3’) were used to amplify the 16S rRNA (rrs) gene frag-
ment specific for A. phagocytophilum. The tick lysates from posi-
tive reactions obtained in our previous investigation [9] served
as a positive control and the double distilled water as a negative
control in each PCR run. All PCR reactions were carried out
in Perkin Elmer GeneAmp PCR System 9700 thermal cyclers.
Amplification products were analyzed after electrophoresis in a
2% agarose gel stained with ethidium bromide. DNA bands of
247 base pairs (bp) were considered positive results. All positive
samples were confirmed with nested PCR reaction amplifying
rrs gene according to Massung [10].
Serological tests
Human
phagocytophilum IgM and IgG antibodies were detected by
indirect immunofluorescence technique applying commercial
kits with HL60 cells infected with the human isolate of A. phago-
granulocytic anaplasmosis (HGA) Anti-A.
cytophilum (Focus technologies HGE IFA IgG /IgM Test Kit,
USA). The serum screening dilution was 1:64 according to the
manufacturer and results 1:64 were considered positive.
Lyme borreliosis In order to examine the anti-Borrelia
burgdorferi serological response Borrelia recombinant IgG and/
/or IgM kits (Biomedica, Austria) applying MiniBoss were used,
according to the producer.
Statistics
Statistical analysis was performed with Pearson’s
p 0.05 was considered significant.
2 test and
Results
Ticks
Of the 372 ticks examined, 54 (14.5%) yield the posi-
tive PCR reaction. The highest prevalence was detected in
females, up to 27.8% (37/133), almost one third lower in males
– 9.2% (13/142), followed by nymphs – 4.1% (4/97). The overall
infection rate varied depending on the collection site; among
adults from 27.3% in Dzikie to 11.8% in Białystok-Pietrasze;
differences in nymphs were less pronounced – from 0% to
8.3%.
Human seropositivity
IgG antibodies against A. phagocytophilum were detected
in 9 out of 231 individuals (3.9%), Tab. 2. A. phagocytophilum
antibodies were detected in employee from 3 forest inspector-
ates – Supraśl 2/36 (5.5%), Dojlidy 3/39 (7.7%) and Czarna
Białostocka 4/72 (5.6%). No seropositive individuals were found
neither in ednia 0/30 nor in Waliły 0/45 inspectorates. Such
a low prevalence of specific antibodies might not allow finding
any significant differences depending on sex, age, length and
character of forestry employment – field or office work, Lyme
borreliosis serological status and history (erythema migrans),
and actually reported complains (fever, arthralgia, mialgia).
Surprisingly, no significant differences were detected between
individuals reporting tick bites in the last year – 6/147 (2.6%)
and those denying it – 3/63 (1.3%). Thus regression analysis was
Table 1. Anaplasma phagocytophilum infection rate in host seeking Ixodes ricinus ticks collected in different forested areas in north-eastern
Poland
Collection siteYear
No positive I. ricinus/No tested (% positive)
MaleTotal adults
4/49 (8.2)10/85 (11.8)
0/4
5/24 (20.8) 13/52 (25.0)
3/27(11.1)*15/59 (25.4)#
0/4 nc2/11 (18.2)
1/28 (3.6) 6/49 (12.2)
0/5 nc3/11 (27.3)
0/1 nc
13/142 (9.2)*50/275 (18.1)#
Female
6/36 (16.6)
1/3 nc
8/28 (28.5)
12/32 (40.0)*
2/7 nc
5/21 (23.8)
3/6 nc
-
37/133 (27.8)*
Nymphs
-
-
1/31 (3.2)
1/23 (4.3)#
0/2 nc
2/24 (8.3)
0/11
0/6 nc
4/97 (4.1)#
Total
Białystok – Pietrasze
Białystok – Strzelnica
Supraśl – Pólko
Królowy Most
Bobrowa
Korytne
Dzikie
Biebrzański PN
2003
2001
2006
2006
2001
2001
2001
2002
10/85 (11.8)
1/7 nc
14/83 (16.8)
16/82 (19.5)
2/13 (15.4)
8/73 (11.0)
3/22 (13.6)
0/7 nc
54/372 (14.5)
1/7 nc
0/1 nc
Total
nc – not calculated, No<10; * – difference statistically significant between females and males; # – difference statistically significant between adults
and nymphs

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Anaplasma phagocytophilum in Ixodes ricinus ticks and human granulocytic anaplasmosis seroprevalence among forestry rangers in Białystok region
not possible. IgM antibodies against A. phagocytophilum were
not detected in any person evaluated.
Lyme borreliosis
Sixty-seven individuals 67/231 (29%) reported erythema
migrans in their medical history. Twelve foresters had erythema
migrans in the year of examination, but only 3 out of 10 persons
tested had IgM antibodies to B. burgdorferi. IgM anti-Borrelia
burgdorferi antibodies were detected in 32 out of 121 (26.4%)
persons tested, IgG – in 43 out of 231 (18.6%), however, the
results were not confirmed by Western Blot tests, Tab. 2.
Discussion
Our results show pretty high A. phagocytophilum infection
rate in I. ricinus ticks collected in north-eastern Poland, reach-
ing 18.1% average in adults and even 40% among females in
certain locations (Królowy Most). The frequency of infection
significantly raises from nymphal to adult stage of ticks. This
observation points to the role of little rodents and small,
and intermediate mammals, harboring I. ricinus larvae, in
A. phagocytophilum circulation in nature since this bacterium is
not transovarially transmitted [1,4]. A. phagocytophilum infec-
tion rate demonstrated in our study is similar to that found in
mid-eastern Poland [11]. Slovac research conducted in suburban
forest near Košice revealed 12.5% adult ticks infected with
A. phagocytophilum [12]. Several other Polish studies showed
the A. phagocytophilum infection rates ranging from 0% in
Szczecin province (Głębokie Public Bath and Landscape Park
in Ińsko) [13], 13.1% in Lublin province [11], 14% in Tricity For-
est on the Baltic coast [14] and to 16% in Białowiea Primeval
Forest [15].
Despite the high A. phagocytophilum infection rate in I. rici-
nus ticks, a very low presence of antibodies (3.9%) was revealed
in the present study. Beginning the investigation among forestry
workers and choosing the summer months, which followed
the highest tick activity, higher prevalence and eventually
acute granulocytic anaplasmosis cases detection was expected.
Other Polish studies in forestry rangers demonstrated higher
levels of seropositivity from 17.7% (20/113) to 20.0% (13/63) in
mid-eastern Poland and 9.6% (46/478) in northern and north-
-eastern Poland [11,16,17]. Analogically to our results, very low
A. phagocytophilum seropositivity – 1.5% was detected in Eng-
lish farmers [18], nevertheless south-western German data from
Baden-Württemberg forestry workers show seroprevalence
ranging from 5% to 16% in various counties [19]. Northern
Italy investigation showed 8.8% (16/181) sera positive by IFA,
although the authors considered only one of them – 0.6%, truly
positive since confirmed by Western blot [20]. However, applica-
tion of Western blot is not required for granulocytic anaplasmo-
sis diagnosis according to ESCMID Study Group [21]. All three
above studies demonstrated anti-B. burgdorferi seropositivity
surpassing those of A. phagocytophilum from 3 to 20 times [11,
19,20], however, the anti-B. burgdorferi assays results should
be interpreted with caution since they where not followed by
Western blot, what is required for Lyme borreliosis diagnostics
[21]. The reasons of such low levels of A. phagocytophilum sero-
positivity are unclear. One of the factors may be very variable
year to year tick infection rate observed in our region during 4
years period [22]. Another factor, postulated by Massung and
co-workers in USA, is presence of A. phagocytophilum variants
non-pathogenic for humans [23], however, the latter hypothesis
require closer characterization of strains postulated.
The results obtained show relatively high A. phagocy-
tophilum infection rate in I. ricinus ticks and very low seropo-
sitivity among forestry rangers, a professional group highly
exposed to ticks.

Acknowledgements
I would like to thank Dr Joanna Stańczak from Medical
Academy of Gdańsk, Institute of Maritime and Tropical Medi-
cine for her help in conducting the above study.
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