Detection of Anaplasma phagocytophilum DNA in Ixodes ticks (Acari: Ixodidae) from Madeira Island and Setubal District, mainland Portugal.

Abstract

A total of 278 Ixodes ticks, collected from Madeira Island and Setubal District, mainland Portugal, were examined by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum. Six (4%) of 142 Ixodes ricinus nymphs collected in Madeira Island and 1 nymph and 1 male (2%) of 93 I. ventalloi collected in Setubal District tested positive for A. phagocytophilum msp2 genes or rrs. Infection was not detected among 43 I. ricinus on mainland Portugal. All PCR products were confirmed by nucleotide sequencing to be identical or to be most closely related to A. phagocytophilum. To our knowledge, this is the first evidence of A. phagocytophilum in ticks from Setubal District, mainland Portugal, and the first documentation of Anaplasma infection in I. ventalloi. Moreover, these findings confirm the persistence of A. phagocytophilum in Madeira Island's I. ricinus.

Full text

Page 1

A total of 278 Ixodes ticks, collected from Madeira
Island and Setúbal District, mainland Portugal, were exam-
ined by polymerase chain reaction (PCR) for the presence
of Anaplasma phagocytophilum. Six (4%) of 142 Ixodes
ricinus nymphs collected in Madeira Island and 1 nymph
and 1 male (2%) of 93 I. ventalloi collected in Setúbal
District tested positive for A. phagocytophilum msp2 genes
or rrs. Infection was not detected among 43 I. ricinus on
mainland Portugal. All PCR products were confirmed by
nucleotide sequencing to be identical or to be most closely
related to A. phagocytophilum. To our knowledge, this is the
first evidence of A. phagocytophilum in ticks from Setúbal
District, mainland Portugal, and the first documentation of
Anaplasma infection in I. ventalloi. Moreover, these findings
confirm the persistence of A. phagocytophilum in Madeira
Island’s I. ricinus.
A
ehrlichiosis agent [HGE agent] [1]) is well established as a
worldwide tickborne agent of veterinary importance and is
considered an emerging human pathogen. The initial
reports of human disease caused by A. phagocytophilum,
now called human granulocytic anaplasmosis, came from
Minnesota and Wisconsin in 1994 (2,3). Human granulo-
cytic anaplasmosis is an acute, nonspecific febrile illness
characterized by headache, myalgias, malaise, and hema-
tologic abnormalities, such as thrombocytopenia and
leukopenia as well as elevated levels of hepatic transami-
naplasma phagocytophilum (formerly Ehrlichia
phagocytophila, E. equi, and the human granulocytic
nases (4). Since that first report, an increasing number of
cases have been described, mostly in the upper Midwest
and in the Northeast regions of the United States (5). Three
years later, in 1997, acute cases of this disease were also
described in Europe (6,7). Several serologic and poly-
merase chain reaction (PCR)-based studies described the
wide distribution of A. phagocytophilum across Europe
and in some parts of the Middle East and Asia (8–10).
Nevertheless, confirmed cases of human granulocytic
anaplasmosis are rare; most European cases are described
in Slovenia (11), with only a few reports from other
European countries (12) and China (13).
The ecology of A. phagocytophilum is still being
defined, but the agent is thought to be maintained in nature
in a tick-rodent cycle, similar to that of Borrelia burdgdor-
feri (the agent of Lyme disease), with humans being
involved only as incidental “dead-end” hosts (14–17).
Exposure to tick bites is considered to be the most com-
mon route of human infection, although human granulo-
cytic anaplasmosis has been reported after perinatal
transmission or contact with infected animal blood
(18,19). A. phagocytophilum is associated with Ixodes
ticks that are known vectors, including I. scapularis, I.
pacificus, and I. spinipalpis in the United States
(15,20,21), I. ricinus mostly in southern, central and north-
ern European regions (22–26), I. trianguliceps in the
United Kingdom (27), and Ixodes persulcatus in eastern
parts of Europe (28) and Asia (9).
In Portugal little information is available concerning
the epidemiology of A. phagocytophilum; the agent was
documented only once in I. ricinus ticks from Madeira
Island (Núncio MS, et al, unpub data). However, the true
prevalence and public health impact of A. phagocytophilum
Detec tion of Anaplasma
phagocytophilum DNA in Ixodes
T icks (Ac ari: Ixodidae) from
Madeira Island and S etúbal Distric t,
Mainland Portugal
Ana Sofia Santos,* Maria Margarida Santos-Silva,* Victor Carlos Almeida,† Fátima Bacellar,*
and John Stephen Dumler‡
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 9, September 2004 1643
*Instituto Nacional de Saúde Dr. Ricardo Jorge, Águas de Moura,
Portugal; †Direcção Regional de Pecuária, Funchal, Portugal; and
‡Johns Hopkins University School of Medicine, Baltimore,
Maryland, USA

Page 2

is likely underestimated since little research has been con-
ducted on this bacterium in Portugal. In fact, seasonal out-
breaks of enzootic abortions and unspecific febrile illness
(commonly named pasture fever) in domestic ruminants,
which could be attributable to A. phagocytophilum, have
been known to breeders and veterinarians across the coun-
try for years. Thus, to expand knowledge of A. phagocy-
tophilum in Portugal, a detailed investigation was initiated.
The preliminary results concerning agent distribution are
presented here. The purpose of this study was to investigate
both the persistence of A. phagocytophilum on Madeira
Island, where it was initially described, and the presence of
the agent in Ixodes ticks from mainland Portugal.
Materials and Methods
Tick Sampling
During 2003 and the beginning of 2004, adults and
nymphs were collected from one site on Madeira Island
(site 1, Paúl da Serra–Porto Moniz) and from five different
sites in the Setúbal District, mainland Portugal (site 2,
Barris–Palmela; site 3, Baixa de Palmela; site 4,
Picheleiros–Azeitão, site 5, Azeitão, site 6, Maçã–
Sesimbra) (Figure 1). Most ticks were unfed, actively
questing arthropods; they were obtained by flagging vege-
tation on pastures and wooded areas bordering farms and
country houses. In site 3, additional specimens were also
collected from domestic cats (Felis catus domesticus). The
ticks were identified by morphologic characteristics
according to standard taxonomic keys (29,30).
Preparation of DNA Extracts from Ticks
Ticks were processed individually as described (25).
Briefly, each tick was taken from the 70% ethanol solution
used for storage, air dried, and boiled for 20 min in 100 µL
of 0.7 mol/L ammonium hydroxide to free DNA. After
cooling, the vial with the lysate was left open for 20 min at
90°C to evaporate the ammonia. The tick lysate was used
directly for PCR. To monitor for occurrence of false-posi-
tive samples, negative controls were included during
extraction of the tick DNA(one control sample for each six
tick samples, with a minimum of two controls).
PCR Amplification
DNA amplifications were performed in a Biometra T-3
thermoblock thermal cycler (Biometra GmbH, Göttingen,
Germany) with two sets of primers: msp465f and msp980r,
derived from the highly conserved regions of major sur-
face protein-2 (msp2) paralogous genes of A. phagocy-
tophilum (31), and ge9f and ge10r, which amplify a
fragment of the 16S rRNA gene of A. phagocytophilum
(3). PCR was performed in a total volume of 50 µL that
contained 1 µmol/L of each primer, 2.5 U of Taq DNA
polymerase (Roche, Mannheim, Germany), 200 µmol/L of
each deoxynucleotide triphosphate (GeneAmp PCR
Reagent Kit, Perkin-Elmer, Foster City, CA), 10 mmol/L
Tris HCL, 1.5 mmol/L MgCl2, and 50 mmol/L KCl pH 8.3
(Roche), as described (3,31). Adult ticks were tested indi-
vidually by using 5 µL of DNA extract. Nymphs were
pooled according to geographic site, up to a maximum of
10 different tick extracts per reaction, and 10 µL of the
pooled DNA was used for initial screening. All positive
pools were confirmed in a second PCR round that used 5
µL of original DNA extract from each nymph. PCR prod-
ucts were separated on 1.5% agarose by electrophorectic
migration, stained with ethidium bromide, and visualized
under UV light. Quality controls included both positive
and negative controls that were PCR amplified in parallel
with all specimens. To minimize contamination, DNA
preparation with setup, PCR, and sample analysis were
performed in three separate rooms.
DNA Sequencing and Data Analysis
Each positive PCR product was sequenced after DNA
purification by a MiniElute PCR Purification Kit (Qiagen,
Valencia, CA). For DNA sequencing, the BigDye termina-
tor cycle sequencing Ready Reaction Kit (Applied
Biosystems, Foster City, CA), was used as recommended
by the manufacturer. Sample amplifications were per-
formed with the forward and reverse primers used for PCR
identification (3,31), with the following modifications: 25
cycles of 96°C for 10 s, 4°C below the melting temperature
of each primer for 5 s, and 60°C for 4 min. Dye Ex 96 Kit
(Qiagen) was used to remove the dye terminators.
Sequences were determined with a 3100 Genetic Analyzer
sequencer (Applied Biosystems). After review and editing,
sequence homology searches were made by BLASTN
1644Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 9, September 2004
RESEARCH
Figure 1. Collection sites in Madeira Island and Setúbal District,
mainland Portugal. S, collection site.

Page 3

analysis of GenBank. Sequences were aligned by using
ClustalX (32) with the neighbor-joining protocol and
1,000 bootstrap replications, and comparing with the 2
msp2 paralogs of A. phagocytophilum Webster strain
(AY253530 and AF443404), one msp2 paralog of USG3
strain (AF029323), and with A. marginale msp2
(AY138955) and msp3 (AY127893) as outgroups.
Dendrograms illustrating the similarity of msp2s were
visualized with TreeView (33).
Results
A total of 278 Ixodes ticks were tested for A. phagocy-
tophilum DNA, including 142 I. ricinus from Madeira
Island and 43 I. ricinus and 93 I. ventalloi from Setúbal
District. The site of collection, origin, and tick stage are
shown in Table 1 and Figure 1. PCR performed with the
msp2 primers detected A. phagocytophilum DNA in seven
pools of nymphs (six pools of 10 I. ricinus from site 1,
Madeira Island, and one pool of 4 I. ventalloi from site 3,
Setúbal District) and also in 1 male I. ventalloi from site 3,
Setúbal District, as demonstrated by the characteristic 550-
bp band. PCRs conducted on individual ticks that com-
prised positive pools confirmed the results and showed that
only one nymph per positive pool contained A. phagocy-
tophilum DNA(Tables 1 and 2). PCR test results were neg-
ative for all I. ricinus collected in the sites in Setúbal
District. Overall, the infection rate was 6 (4%) of 142 for
I. ricinus and 2 (2%) of 93 for I. ventalloi. Analysis based
on direct amplicon sequencing showed the expected con-
served 5′ end followed by ambiguous sequences that cor-
responded to the hypervariable central region of msp2, as
anticipated based on the presence of >52 msp2 copies in
the A. phagocytophilum HZ strain genome (34). Thus, for
appropriate comparison and alignment, the msp2 5′
sequences were edited from the positions where unam-
biguous reads could be determined and terminated 70 nt
into the sequence at the approximate beginning of the
hypervariable region. A similar alignment protocol for the

Table 1. Results of PCR to detect Anaplasma phagocytophilum DNA in ticks
3′ end of the msp2 amplicons showed more ambiguous
positions, which prohibited effective alignment and
sequence determination. Thus, msp2 sequence alignments
depended upon approximately 70 nt 5′ to the hypervariable
region and were performed less for phylogenetic stratifica-
tion of A. phagocytophilum in the ticks than to confirm that
the amplified msp2 sequences were not derived from other
related Anaplasma or Ehrlichia spp. The nucleotide
sequences determined for this 70-bp region amplified from
all eight ticks showed 98.5%–85.7% similarity,
94.2%–86.9% similarity when compared to representative
msp2 sequences of A. phagocytophilum Webster and
USG3 strains, and 63.7%–35.0% similarity when com-
pared to A. marginale msp2 and msp3 sequences (Figure
2). Sequences obtained from the two I. ventalloi from
mainland Portugal clustered together and separately from
other msp2 sequences obtained from I. ricinus on Madeira
Island (Figure 2).
When amplified by using rrs primers ge9f and ge10r,
compared to A. phagocytophilum U02521, sequences were
99% identical to two I. ventalloi (636/640 positions and
846/848 positions, respectively) on mainland Portugal and
to three I. ricinus (836/841, 817/820, and 838/839 posi-
tions, respectively) on Madeira Island.
Discussion
This study constitutes part of a larger effort to investi-
gate the distribution of A. phagocytophilum in various
regions of Portugal. Our data provide supporting evidence
that A. phagocytophilum is present in actively questing I.
ricinus from Madeira Island and in I. ventalloi from
Setúbal District, mainland Portugal.
We used two approaches for identifying A. phagocy-
tophilum in ticks: 1) standard amplification of rrs that can
have limited sensitivity because of a single copy in each
bacterial genome, and 2) amplification of msp2, a gene for
which as many as 52 paralogs are present in the A. phago-
cytophilum genome and for which detection sensitivity is
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 9, September 2004 1645
Anaplasma phagocytophilum in Portuguese Ticks
a
Ixodes ricinus
Nymphs

6/139

0/1
0/2
I. ventalloi
b
Area
Madeira Island
Paúl da Serra–Porto Moniz
Setúbal District Portugal Mainland
Barris–Palmela
Baixa de Palmela
Site

1

2
3
Origin

Vegetation

Vegetation
Vegetation
Felis catus
domesticus
Vegetation
Vegetation
Vegetation

b
F
b
M
b
NymphsF
b
M
b
Total
c

0/2

0/5
0/2
0/1

0/7
0/2
–

–
–

–
–

142

14
34
0/1
0/7 1/15 0/6
–
–
–
–
–
0/2
–
0/10
21
–
0/2
0/1
0/9
22
– 0/6
0/9
–
0/4
25
1/4
0/18
0/1
0/9
40
10
43
2
33
278
Picheleiros–Azeitão
Azeitão
Maçã–Sesimbra
Total
aPCR, polymerase chain reaction; F, female; M, male.
bNumber of positives ticks/number of ticks examined.
cTotal number of ticks examined.
4
5
6

0/12
–
0/1
28
c
142

Page 4

enhanced (34). The pitfall of msp2 amplification derives
from targeting conserved sequences that flank a hypervari-
able central region, which results in amplicons with partial
sequence ambiguity when cloning is not attempted before
sequencing (31). These findings are highly unlikely to rep-
resent amplicon contamination since marked sequence
diversity was observed, and since only a single tick from
each pool was positive in each reaction. Although only
limited data can gleaned by this analysis, which interro-
gates only nucleic acids of small size, Casey et al. have
shown that msp2 “similarity” groups, reflecting clusters
determined by a similar sequencing approach, can be use-
ful in predicting phylogenetic relationships, particularly
with reference to adaptation to specific host niches (35).
Madeira, the main island of the Madeira Archipelago, is
located in the North Atlantic Ocean, about 800 km west of
the African continent and 1,000 km from the European
coast. On this island, I. ricinus is the most abundant tick
species and the only Ixodes tick that was found in this
study. A. phagocytophilum was detected in 4% of I. ricinus
collected in Paúl da Serra. Our results corroborate previous
findings, although prevalence here is slightly lower than
the 7.5% infection rate in ticks previously collected in sim-
ilar areas (Núncio MS, et al., unpub data). These differ-
ences may be attributable to seasonal variations in A.
phagocytophilum prevalence within reservoir hosts or
ticks or to technical aspects of detection. Regardless, stud-
ies that use a greater number of samples and that are per-
formed in different seasons, locations, and habitats will be
needed to confirm the levels of infection. Nevertheless,
these findings are generally similar to those described else-
where in Europe, although prevalence rates can vary great-
ly with the origin of I. ricinus examined, ranging from a
minimum of <1% in the United Kingdom, France, and
Sweden (23,24,36) to a maximum of 24% to 29% in north-
ern Italy, Germany, and Spain (22,25,26). The public
health importance of these findings still remains to be
determined. I. ricinus is an exophilic, three-host tick
known to bite several domestic animals and humans in
Portugal (30). Therefore, we can assume that the presence
of A. phagocytophilum on Madeira Island I. ricinus sug-
gests a potential health threat to animals and humans and
should be investigated.
Mainland Portugal is the most western region of
Europe, with an area of 89,000 km2, divided into 18 dis-
tricts. Although I. ricinus is not the main tick species in
mainland Portugal, it can be found across the country in
habitats with favorable conditions. Focused in Setúbal
District, to the south of the Tejo River, our study detected
I. ricinus in all five sites chosen for field work: Barris;
Baixa de Palmela; Picheleiros; Azeitão, and Maçã. In those
sites, the distribution of I. ricinus was accompanied by
another Ixodes species, I. ventalloi. Another ecologically
interesting finding that should be further confirmed was
that, although all of the I. ricinus from mainland Portugal
tested negative, evidence of A. phagocytophilum was
found in 2% of all I. ventalloi, including 5% collected in
Baixa de Palmela. The msp2 sequences identified in these
two ticks were more closely related to each other than to
any msp2 sequence identified in ticks from Madeira Island.
In contrast, A. phagocytophilum msp2 diversity in I. rici-
nus from Madeira Island was broad and showed overlap
with gene sequences identified in North American strains,
1646 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 9, September 2004
RESEARCH
Table 2. PCR-positive results of ticks
Sites
Madeira Island
1
Setúbal District Mainland Portugal
3
aPCR, polymerase chain reaction.
a
No. positive nymphs

6

1
No. positive adults

–

1
Tick extracts codes

11; 60; 93; 118; 122; 137

160; 246 (respectively)
Figure 2. Dendrogram showing the phylogenetic relationships of
the msp2 sequences of the newly identified strains and other rep-
resentative sequences from North American Anaplasma phagocy-
tophilum strains (Webster strain–Wisconsin and USG3
strain–eastern United States), and from A. marginale Florida strain
(msp2 and msp3). Bootstrap values (out of 1,000 iterations) are
shown at the nodes. Bar, substitutions/1,000 bp.

Page 5

as observed for some A. phagocytophilum strains in the
United Kingdom (35).
To our knowledge, this identification of A. phagocy-
tophilum in ticks is the first from mainland Portugal and
the first documentation of Anaplasma infection in I. ven-
talloi. This species is an endophilic, three-host tick well
adapted to a broad range of habitats that vary from open,
dry forest in semidesert Mediterranean areas to the mild
humid conditions in the southern part of the British Isles.
In Portugal, I. ventalloi infest a variety of small rodents,
carnivores, and lizards but have not been found to feed on
humans (30). A. phagocytophilum has already been report-
ed in other ticks, besides the known vector species
(37–41). The presence in alternate ticks is attributable to
the existence of secondary maintenance cycles, in which A.
phagocytophilum circulates between relatively host-spe-
cific, usually nonhuman-biting ticks and their hosts
(38,39). Those additional cycles would buffer the agent
from local extinction and help reestablish the primary
cycles (38,39). Although this hypothesis might explain our
results, the competency of I. ventalloi to act as vector for
A. phagocytophilum has yet to be demonstrated. Moreover,
the different average prevalences observed in each location
suggest that A. phagocytophilum is not widely spread in
ticks and that some reservoir animals or hosts are needed
for its maintenance. Trapping and animal surveillance are
needed to provide more information that could help to
explain the biological importance of those findings.
Acknowledgments
We thank Maria Arminda Santos for useful help in field
work and Sonia Pedro from National Health Insitute Ricardo
Jorge for valuable assistance with sequencing.
This research was partially supported by the Portuguese
government through the Fundação para a Ciência e a Tecnologia
grant BD/8610/2002 and through U.S. National Institutes of
Health grant R01-AI41213 to J.S.D.
Ms. Santos works at the Centre of Vector-Borne and
Infectious Diseases, National Institute of Health, Portugal. Her
research has been focused on tickborne agents, mainly rickettsial
agents with human health importance. She is pursuing a doctoral
degree focused on human anaplasmosis and ehrlichiosis in
Portugal.
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Address for correspondence: Ana Sofia Santos, Centro de Estudos de
Vectores e Doenças Infecciosas, Instituto Nacional de Saúde Dr. Ricardo
Jorge, Avenida da Liberdade 5, 2965-575 Águas de Moura, Portugal; fax:
+351-265-912568; email: ana.santos@insa.min-saude.pt
1648 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 9, September 2004
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