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Ticks
and
Tick-borne
Diseases
7
(2016)
1198–1202
Contents
lists
available
at
ScienceDirect
Ticks
and
Tick-borne
Diseases
j
ourna
l
ho
me
pa
ge:
www.elsevier.com/locate/ttbdis
Molecular
detection
and
identification
of
Rickettsiales
pathogens
in
dog
ticks
from
Costa
Rica
Liliana
Campos-Calderóna,
Leyda
Ábrego-Sánchezb,
Antony
Solórzano-Moralesa,
Alberto
Albertic,
Gessica
Torec,
Rosanna
Zobbac,
Ana
E.
Jiménez-Rochaa,
Gaby
Dolza,b,∗
aEscuela
de
Medicina
Veterinaria,
Universidad
Nacional,
Campus
Benjamín
Nú˜
nez,
Barreal
de
Heredia,
Costa
Rica
bMaestría
en
Enfermedades
Tropicales,
Posgrado
Regional
en
Ciencias
Veterinarias
Tropicales,
Universidad
Nacional,
Campus
Benjamín
Nu˜
nez,
Barreal
de
Heredia,
Costa
Rica
cDepartment
of
Veterinary
Medicine,
University
of
Sassari,
Via
Vienna
2,
07100
Sassari,
Italy
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
29
February
2016
Received
in
revised
form
1
July
2016
Accepted
24
July
2016
Available
online
25
July
2016
Keywords:
Rhipicephalus
sanguineus
s.l.
a
b
s
t
r
a
c
t
Although
vector-borne
diseases
are
globally
widespread
with
considerable
impact
on
animal
production
and
on
public
health,
few
reports
document
their
presence
in
Central
America.
This
study
focuses
on
the
detection
and
molecular
identification
of
species
belonging
to
selected
bacterial
genera
(Ehrlichia,
Anaplasma
and
Rickettsia)
in
ticks
sampled
from
dogs
in
Costa
Rica
by
targeting
several
genes:
16S
rRNA/dsb
genes
for
Ehrlichia;
16S
rRNA/groEL
genes
for
Anaplasma,
and
ompA/gltA/groEL
genes
for
Rick-
ettsia.
PCR
and
sequence
analyses
provides
evidences
of
Ehrlichia
canis,
Anaplasma
platys,
and
Anaplasma
phagocytophilum
infection
in
Rhipicephalus
sanguineus
s.l
ticks,
and
allow
establishing
the
presence
of
Rickettsia
monacensis
in
Ixodes
boliviensis.
Furthermore,
the
presence
of
recently
discovered
Mediter-
ranean
A.
platys-like
strains
is
reported
for
the
first
time
in
Central
America.
Results
provide
new
background
on
geographical
distribution
of
selected
tick-transmitted
bacterial
pathogens
in
Costa
Rica
and
on
their
molecular
epidemiology,
and
are
pivotal
to
the
development
of
effective
and
reliable
diag-
nostic
tools
in
Central
America. ©
2016
Elsevier
GmbH.
All
rights
reserved.
1.
Introduction
In
tropical
regions,
tick-
transmitted
infections
are
an
emerg-
ing
problem
in
dogs,
causing
serious
disease
or
sub
clinical
infections
making
companion
animals
a
reservoir
for
human
vector-transmitted
infectious
agents,
since
most
of
tick-borne
bac-
terial
agents
that
infect
dogs
are
zoonotic
(Nicholson
et
al.,
2010).
The
location
of
Costa
Rica
in
the
neotropic
ecozone
provides
the
ideal
conditions
for
the
establishment
and
development
of
a
variety
of
ticks.
Most
bacterial
tick-borne
pathogens
have
been
described
to
cause
disease
in
dogs
(Day,
2011):
Ehrlichia
canis,
Anaplasma
phago-
cytophilum,
and
Rickettsia
rickettsii
(Carrade
et
al.,
2009;
Little
et
al.,
2010;
Nicholson
et
al.,
2010).
The
brown
dog
tick
Rhipicephalus
sanguineus
sensu
lato
(s.l.)
(Moraes-Filho
et
al.,
2011)
is
the
most
∗Corresponding
author
at:
Escuela
de
Medicina
Veterinaria,
Universidad
Nacional,
P.O.
Box
80-3000
Heredia,
Costa
Rica.
E-mail
addresses:
lilliana.campos.calderon@una.cr
(L.
Campos-Calderón),
leabrego@hotmail.com
(L.
Ábrego-Sánchez),
antony.solorzano.morales@una.cr
(A.
Solórzano-Morales),
alberti@uniss.it
(A.
Alberti),
rosannazobba@uniss.it
(R.
Zobba),
anajimenez@racsa.co.cr
(A.E.
Jiménez-Rocha),
gaby.dolz.wiedner@una.cr
(G.
Dolz).
common
tick
reported
in
dog
infestations
in
Costa
Rica.
Amblyomma
cajennense
(reported
as
Amblyomma
mixtum),
Amblyomma
macu-
latum,
Amblyomma
ovale,
Amblyomma
pecarium,
Ixodes
boliviensis
and
Rhipicephalus
microplus
have
been
found
sporadically
on
the
same
host
species
(Álvarez
et
al.,
2005,
2006;
Troyo
et
al.,
2012).
Innovative
molecular
methods
have
been
combined
to
develop
use-
ful,
sensitive,
and
rapid
tools
for
the
detection
and
identification
of
vector-borne
pathogens
in
arthropods,
including
ticks
(Nicholson
et
al.,
2010).
Although
there
are
several
reports
about
prevalence
and
molecular
detection
of
Rickettsiales
agents
in
dogs
(Abrego
et
al.,
2009;
Romero
et
al.,
2010,
2011;
Rojas
et
al.,
2014;
Troyo
et
al.,
2014),
to
date,
findings
in
ticks
from
Costa
Rica
are
still
lack-
ing.
This
study
reports
the
molecular
detection
and
characterization
of
selected
species
of
the
order
Rickettsiales
(Ehrlichia,
Anaplasma,
and
Rickettsia)
in
ticks
collected
from
dogs
of
Costa
Rica.
2.
Material
and
methods
2.1.
Collection
and
identification
of
ticks
A
prospective
observational
convenience
study
was
carried
out
in
order
to
enroll
dogs
attended
by
veterinarians.
After
open
invi-
http://dx.doi.org/10.1016/j.ttbdis.2016.07.015
1877-959X/©
2016
Elsevier
GmbH.
All
rights
reserved.
L.
Campos-Calderón
et
al.
/
Ticks
and
Tick-borne
Diseases
7
(2016)
1198–1202
1199
tation,
14
veterinary
clinics
gave
their
consent
to
participate
on
voluntary
basis
in
the
investigation.
Clinics
were
located
mainly
in
the
Great
Metropolitan
Area
(GMA)
of
the
country
(San
José,
Ala-
juela,
Cartago
and
Heredia
provinces),
and
some
in
the
Guanacaste
province
in
the
north
Pacific
coast
of
Costa
Rica.
During
October
2006
to
July
2007,
all
ticks
observed
in
dogs
presented
at
the
clin-
ics
or
attended
by
veterinarians
in
residences
were
collected
and
deposited
in
plastic
tubes
containing
70%
alcohol.
Identification
of
ticks
at
species
level
was
performed
morphologically
using
a
stereoscope
and
the
taxonomic
keys
proposed
by
Fairchild
et
al.
(1966)
and
Barros-Battesti
et
al.
(2006).
Identification
of
nymphs
was
carried
out
according
to
Cooley
(1946).
2.2.
DNA
extraction
and
polymerase
chain
reaction
(PCR)
DNA
was
extracted
from
ticks
using
DNeasy®Blood
&
Tissue
Kit
(QIAGEN,
Chatsworth,
CA,
USA),
by
following
the
manufac-
turer’s
instructions.
All
ticks
(adults
and
nymphs)
were
individually
treated.
A
set
of
already
published
primers
was
selected
and
used
to
identify
the
Rickettsiales
bacterial
species
most
frequently
reported
in
dogs
(Table
1).
Briefly,
the
presence
of
bacterial
species
belonging
to
the
fam-
ily
Anaplasmataceae
and
Rickettsiaceae
was
investigated
with
primers
targeting
the
16s
rRNA
and
ompA/gltA/groEL
genes,
respec-
tively.
In
order
to
mine
deeper
into
Anaplasmataceae
diversity
specific
PCR
assays
were
conducted
to
detect
E.
canis,
Ehrlichia
chaffeensis,
and
Ehrlichia
ewingii
(nested
16S
rRNA
and
dsb);
A.
phagocytophilum
(nested
16S
rRNA
and
groEL),
and
A.
platys/A.
platys-like
(nested
16S
rRNA
and
groEL).
DNA
from
A.
phago-
cytophilum
(strain
Trestom)
HL-60
infected
cells
was
used
as
positive
control
for
A.
phagocytophilum
PCR
detection;
similarly,
DNA
extracted
from
blood
of
a
dog
positive
by
PCR
to
A.
platys
(and
confirmed
by
sequencing)
was
used
as
positive
control
during
detection
of
A.
platys
(Abrego
et
al.,
2009).
Plasmids
containing
seg-
ments
of
DNA
from
E.
canis,
E.
chaffeensis
and
E.
ewingii
were
used
as
positive
controls
in
Ehrlichia
PCR
experiments,
whereas
Rickettsia
felis
DNA
was
used
as
positive
control
for
Rickettsia
spp.
All
PCR
experiments
included
water
(Fermentas®)
used
as
negative
con-
trol.
PCR
products
were
visualized
by
agarose
gel
electrophoresis
(1.4%)
in
TBE
(Tris
Base,
boric
acid,
EDTA,
pH8,
0.5
M),
and
ethidium
bromide
staining
(0.5
g/ml).
GeneRuler
100
bp
DNA
Ladder
Plus
(Sm0321,
Fermentas®)
was
used
for
DNA
sizing.
2.3.
Sequencing
and
sequence
analysis
In
order
to
confirm
PCR
results,
amplicons
obtained
with
primers
specifically
targeting
A.
phagocytophilum
(16S
rRNA
and
groEL),
A.
platys
(16S
rRNA
and
groEL),
Rickettsia
spp.
(gltA
and
groEL),
or
the
ehrlichial
dsb
gene,
were
sequenced
and
compar-
ative
sequences
analyses
were
performed.
Shortly,
PCR
products
were
purified
using
the
QIAquick®(QIAGEN)
kit,
by
following
the
manufacturer’s
instructions.
One
E.
canis
sample
(dsb),
two
A.
phagocytophilum
samples
(16S
rRNA
and
groEL),
one
A.
platys
sample
(16S
rRNA
and
groEL),
one
A.
platys-like
sample
(groEL),
one
sample
positive
to
Rickettsia
spp.
(gltA
and
groEL),
were
automatically
sequenced
by
commercial
companies.
Sequences
were
aligned
with
the
ClustalW
option
of
BioEdit
(Hall,
1999)
and
compared
with
sequences
deposited
in
the
NCBI
database
(National
Center
for
Biotechnology
Information)
using
the
BLASTn
algorithm.
3.
Results
A
total
of
165
ticks
were
collected
from
165
dogs
that
were
either
attended
at
veterinary
clinics
(157)
or
in
residences
(8),
of
which
79
came
from
the
province
of
Heredia,
53
from
Alajuela,
21
from
San
Jose,
8
from
Cartago,
2
from
Guanacaste
(province
at
the
Pacific
coast,
outside
the
GMA),
and
2
from
undefined
locations.
One
hundred
fifty-six
were
adult
ticks
(104
females
and
52
males)
whereas
9
were
nymphs.
The
165
ticks
were
identified
as
R.
san-
guineus
s.l.
(n
=
160),
A.
cajennense
complex
(n
=
4),
and
I.
boliviensis
(n
=
1).
Upon
an
initial
screening
conducted
with
primers
specific
for
Anaplasmataceae,
14
out
of
165
DNA
samples
(8.5%)
tested
positive
(Table
2).
Subsequently,
more
specific
and
sensitive
PCR
meth-
ods
were
applied
in
order
to
mine
deeper
into
the
presence
of
Anaplasma
and
Ehrlichia
species.
E.
canis
was
detected
in
43
(26%)
R.
sanguineus
s.l.
ticks
(Table
2),
of
these,
5
were
nymphs
and
38
were
adults
(22
females
and
16
males).
The
highest
proportion
of
E.
canis
positive
ticks
was
found
in
the
provinces
of
Heredia
and
Alajuela
(Table
3)
although
positivity
was
detected
in
all
the
provinces
of
the
Costa
Rican
Central
Valley.
On
Blast
search
the
sequence
of
the
E.
canis
dsb
gene
obtained
in
this
study
(KU534872)
resulted
100%
(288/288bp)
similar
to
several
E.
canis
sequences
deposited
in
the
GenBank
detected
in
ticks
of
Argentina
and
Brazil
(KR909452,
KP167596),
and
in
human
blood
donors
of
Costa
Rica
(KR732921).
The
use
of
a
16S
rRNA
A.
phagocytophilum
specific
PCR
allowed
identifying
this
pathogen
in
2
(1.3%)
adult
female
R.
sanguineus
s.l.
ticks
from
Heredia
and
San
Jose
(Table
3).
After
sequencing,
one
of
the
sequences,
designated
as
16S
AphagoCR1
and
deposited
in
GenBank
under
KU534874,
was
found
100%
(343/343
pb)
sim-
ilar
to
A.
phagocytophilum
strains
isolated
in
different
countries
and
different
hosts.
The
second
sequence,
named
16S
AphagoCR2
and
deposited
in
GenBank
under
KU534875,
shared
99.7%
homol-
ogy
(310/311pb)
with
the
same
strains.
When
these
two
ticks
were
analyzed
by
A.
phagocytophilum
specific
groEL
PCR,
only
one
sample,
from
which
sequence
16S
AphagoCR1
was
obtained,
gen-
erated
a
positive
reaction.
GroEL
sequencing
resulted
in
a
sequence
designated
as
GroEL
AphagoCR1
and
deposited
in
GenBank
under
KU534870,
that
had
100%
(530/530pb)
homology
with
A.
phago-
cytophilum
pathogenic
strains
isolated
in
the
USA
and
in
the
Mediterranean
area
(AF172163
and
AY848750,
respectively).
Using
a
16S
rRNA
A.
platys
specific
PCR
this
pathogen
was
iden-
tified
in
5
(3.0%)
R.
sanguineus
s.l.
tick
samples,
of
which
four
were
adults
(3
females
and
1
male)
and
one
was
a
nymph
(Table
2).
Pos-
itive
samples
came
from
the
provinces
of
Heredia
and
Alajuela
(Table
3).
Sequencing
and
BlastN
analyses
showed
that
the
16S
rRNA
sequence
generated
in
this
study
(KU534873)
was
98.5%
sim-
ilar
to
sequences
representative
of
the
16S
rRNA
gene
of
A.
platys
(AF
156784),
and
of
several
related
strains,
such
as
that
found
in
dromedaries
in
Tunisia
(KC800963)
and
that
described
as
Candida-
tus
Anaplasma
camelii
in
Saudi
Arabia
(KF843823).
The
presence
of
A.
platys
was
further
investigated
by
targeting
the
groEL
gene
from
samples
that
yielded
positive
results
in
the
16S
RNA-PCR
for
A.
phagocytophilum
(n
=
2)
and
16S
RNA-PCR
for
A.
platys
(n
=
4).
Only
DNA
extracted
from
one
R.
sanguineus
s.l.
tick
was
found
positive
to
A.
platys
groEL
PCR,
sequencing
and
homology
search
revealed
that
this
sequence
(KU534871)
was
100%
(476/476
pb)
similar
to
sequence
KC335256,
isolated
from
a
calf
in
the
Mediterranean
area,
belonging
to
a
cluster
of
strains
similar
to
but
different
from
A.
platys,
and
infecting
neutrophil
granulocytes.
Interestingly
this
tick
positive
to
A.
platys–like
by
groEL
PCR
was
also
found
positive
to
A.
phagocytophilum
(sequence
16S
AphagoCR1).
A
total
of
26
(16.2%)
ticks,
25
R.
sanguineus
s.l.
and
1
I.
bolivien-
sis,
tested
positive
to
ompA
and
gltA
Rickettisia
spp.
specific
PCR
(Table
2).
Sequencing
and
homology
revealed
the
presence
of
an
invariable
gltA
(KU529481)
and
groEL
(KX447668)
sequence
in
the
female
I.
boliviensis
tick
from
Heredia,
that
on
BlastN
comparison
was
99.7%
(378/379bp)
and
99.8%
(582/583bp)
similar
to
Rickettsia
monacensis
sp.
nov.
(type
strain,
IrR/MunichT)
from
an
Ixodes
rici-
nus
tick
collected
in
Germany
(AF141906.1),
respectively.
The
groEL
1200
L.
Campos-Calderón
et
al.
/
Ticks
and
Tick-borne
Diseases
7
(2016)
1198–1202
Table
1
Primers
used
in
this
study
for
amplifying
selected
tick-borne
pathogens.
Pathogen
Gen
Primer
(Reference)
Sequences
(5-
3)
Fragment
length
(bp)
Anaplasmataceae
16SrRNA
EHR16SD
EHR16SR
(Inokuma
et
al.,
2000)
GGTACCYACAGAAGAAGTCC
TAGCACTCATCGTTTACAGC
345
Rickettsia
spp.
gltA
Cs78
Cs323
(Labruna
et
al.,
2004)
GCAAGTATCGGTGAGGATGTAAT
GCTTCCTTAAAATTCAATAAATCAGGAT
401
Rickettsia
spp. ompA
Rr190.70p
Rr190.602n
(Regnery
et
al.,
1991)
ATGGCGAATATTTCTCCAAAA
AGTGCAGCATTCGCTCCCCCT
532
Rickettsia
spp.
groEL
RgroMAR2-F
RgroMAR2-R
RgroMAR1-F
RgroMAR1-R
(Chisu
et
al.,
2016)
AAAAGCTCGTGAGCAAATGC
GTGATAACCGTTGAAGAAG
GAGAGATGGAAGCAAGTAC
GAAAGATGGATAGTCGCTGA
508
Ehrlichia
spp.
16SrRNA
ECC
ECB
(Romero
et
al.,
2011)
AGAACGAACGCTGGCGGCAAGC
CGTATTACCGCGGCTGCTGGCA
478
E.
canis
16SrRNA
ECAN5
HE3
(Romero
et
al.,
2011)
CAATTATTTATAGCCTCTGGCTATAGGA
TATAGGTACCGTCATTATCTTCCCTAT
389
E.
chaffeensis
16SrRNA
HE1
HE3
(Romero
et
al.,
2011)
CAATTGCTTATAACCTTTTGGTTATAAAT
TATAGGTACCGTCATTATCTTCCCTAT
390
E.
ewingii 16SrRNA
EE5
HE3
(Romero
et
al.,
2011)
CAATTCCTAAATAGTCTCTGACTATTTAG
TATAGGTACCGTCATTATCTTCCCTAT
392
Ehrlichia
spp.
dsb
Dsb-330
Dsb-728
(Romero
et
al.,
2011)
GATGATGTCTGAAGATATGAAACAAAT
CTGCTCGTCTATTTTACTTCTTAAAGT
409
A.
platys
16SrRNA
8F
1448R
EHR16SR
PLATYS
(Ábrego
et
al.,
2009)
AGTTTGATCATGGCTCAG
CCATGGCGTGACGGGCAGTGTG
TAGCACTCATCGTTTACAGC
GATTTTTGTCGTAGCTTGCTATG
678
A.
phagocytophilum
16SrRNA
Ge3a
Ge10r
Ge9f
Ge2
(Massung
et
al.,
1998)
CACATGCAAGTCGAACGGATTATTC
TTCCGTTAAGAAGGATCTAATCTCC
AACGGATTATTCTTTATAGCTTGCT
GGCAGTATTAAAAGCAGCTCCAGG
546
Anaplasma
spp.
groEL
EphplgroEL
EphplgroEL
(Alberti
et
al.,
2005)
ATGGTATGCAGTTTGATCGC
TCTACTCTGTCTTTGCGTTC
624
A.
platys groEL
EphplgroEL
EplgroEL
(Zobba
et
al.,
2014)
ATGGTATGCAGTTTGATCGC
CATAGTCTGAAGTGGAGGAC
515
A.
phagocytophilum
groEL
EphplgroEL
EphgroEL
(Alberti
et
al.,
2005)
ATGGTATGCAGTTTGATCGC
TTGAGTACAGCAACACCACCGGAA
573
Table
2
Number
of
ticks
positive
for
selected
tick-borne
pathogens
amplifying
different
genes
with
PCR.
Pathogen
16SrRNA+/total
dsb+/total
groEL+/total
gltA+/total
ompA+/total
Anaplasmataceae
14/165
–
–
–
–
E.
canis
43/165
1/1
–
–
–
E.
chaffeensis
0/165
–
–
–
–
E.
ewingii
0/165
–
–
–
–
A.
platys
5/165
–
0/4
–
–
A.
phagocytophilum
2/163
–
1/2
–
–
A.
platys-like
–
–
1/6
–
–
Rickettsiaceae
–
–
1/1
26/160
26/160
sequence
was
99.2%
(376/379bp)
similar
to
R.
monacensis
strain
WB9/Ir
Pavullo
(HM210739.1)
isolated
from
I.
ricinus
from
Italy.
4.
Discussion
R.
sanguineus
s.l.
is
the
most
common
tick
infesting
dogs
of
Costa
Rica
(Álvarez
et
al.,
2005,
2006;
Jiménez-Rocha
et
al.,
2013).
Dogs
are
the
closest
animals
to
humans
and
although
reports
in
the
literature
about
human
bites
by
ticks
are
few
and
interaction
of
R.
sanguineus
s.l.
with
humans
must
be
further
investigated,
parasitism
by
these
ticks
have
been
documented,
indicating
a
potential
risk
for
transmission
of
pathogens
to
humans
(Dantas-
Torres,
2008).
Thus,
the
detection
of
E.
canis,
A.
phagocytophilum,
A.
platys,
and
A.
platys-like
in
this
tick
species
is
of
particular
impor-
tance.
However,
since
ticks
were
collected
directly
from
dogs,
our
results
for
Anaplasmataceae
could
have
originated
from
infected
blood
ingested
by
these
ticks,
and
not
necessarily
from
infected
ticks.
L.
Campos-Calderón
et
al.
/
Ticks
and
Tick-borne
Diseases
7
(2016)
1198–1202
1201
Table
3
Geographical
origin
and
infection
rates
of
ticks.
Province
Ticks
Infection
rates
(N)
E.
canis
A.
platys
A.
phagocytophilum
A.
platys-like
R.
monacensis
Heredia
79
21(26.6%)
1
(1.3%)
1
(0.6%)
1
(1.3%)
1
(0.6%)
Alajuela
53
17(32.0%)
4
(7.5%)
–
–
–
San
José
21
2
(9.5%)
–
1
(0.6%)
–
–
Cartago
8
2
(25%) –
–
–
–
Guanacaste
2
–
–
–
–
–
Not
known
2
1
(50%)
–
–
–
–
Total
165
43
(26%)
5
(3%)
2
(1.2%)
1
(0.6%)
1
(0,6%)
Only
14
out
of
165
DNA
samples
tested
positive
for
Anaplas-
mataceae,
what
can
be
explained
by
the
lower
sensitivity
of
this
PCR
compared
to
more
specific
and
nested
PCRs.
However,
it
is
important
to
notice
that
only
8
samples
were
confirmed
as
E.
canis
positive,
or
E.
canis
and
A.
platys
positive
(2
samples),
whereas
the
remaining
4
samples
yielded
negative
results.
This
may
suggest
the
presence
of
a
novel
bacterial
strains
belonging
to
this
family,
that
can
not
be
detected
with
species-specific
PCRs,
or
the
presence
of
other
bacterial
groups,
not
related
to
ticks.
The
high
infection
rate
of
E.
canis
in
Costa
Rica
in
R.
sanguineus
s.l.
ticks
are
in
accordance
with
the
high
seroprevalence
observed
previously
in
dogs
(Barrantes-González
et
al.,
2013).
Populations
of
R.
sanguineus
s.l.
ticks
from
Central
America
are
included
within
a
group
called
tropical
or
northern
lineage
(Dolz
et
al.,
2015),
which
is
known
to
be
associated
with
the
transmission
of
E.
canis
to
dogs
in
the
tropical
areas
of
South
America
(Moraes-Filho
et
al.,
2015).
DNA
of
A.
phagocytophilum
has
been
reported
in
R.
sanguineus
s.l.
from
Brazil
(Santos
et
al.,
2013),
but
to
date
not
in
Central
America.
In
Costa
Rica
two
possible
human
cases
were
diagnosed
previously
but
based
on
clinical
signs
and
detection
of
granulo-
cytic
morulae
in
blood
smears
(Rojas-Solano
and
Villalobos-Vindas,
2007;
Hernández-de
Mezerville
and
Padilla-Cuadra,
2007).
This
is
in
accordance
with
our
results
that
identified
DNA
of
A.
phagocy-
tophilum.
However,
it
remains
to
establish
which
other
genetic
A.
phagocytophilum
variants
are
present
in
Costa
Rica
(Dugat
et
al.,
2015),
and
if
R.
sanguineus
s.l.
plays
a
role
in
the
transmission
of
this
pathogen
to
humans
and
other
animals.
Infections
with
A.
platys
in
R.
sanguineus
s.l.
have
been
reported
in
different
countries
from
Africa,
Asia,
and
Europe
(Latrofa
et
al.,
2014;
Ramos
et
al.,
2014;
Sanogo
et
al.,
2003;
Yba˜
nez
et
al.,
2012).
The
potential
role
of
this
tick
as
biological
vector
was
suggested
(Harvey
et
al.,
1978;
Simpson
et
al.,
1991;
Ramos
et
al.,
2014),
our
results
obtained
in
this
study
show
additional
evidence.
Finding
A.
platys-like
strains
in
ticks
in
Costa
Rica
are
in
agree-
ment
with
recent
detection
of
this
agent
in
Mediterranean
and
Chinese
ruminants
(Zobba
et
al.,
2014;
Yang
et
al.,
2015),
and
sug-
gest
that
species
diversity
of
Anaplasmataceae
has
yet
to
be
fully
investigated,
and
that
further
studies
are
needed
to
identify
more
species
and
variants
in
the
future.
The
only
rickettsias
reported
to
date
in
I.
boliviensis
were
Candi-
datus
Rickettsia
andaneae,
detected
in
a
tick
from
a
horse
in
Peru
(Blair
et
al.,
2004),
and
an
undescribed
Rickettsia
sp.
found
in
a
dog
tick
in
Costa
Rica
(Troyo
et
al.,
2014).
This
rickettsia
was
named
strain
IbR/CRC,
and
placed
in
the
group
of
R.
monacensis,
but
was
also
found
close
to
an
endosymbiont
of
Ixodes
scapularis
and
other
undescribed
rickettsiae.
Our
results
using
gltA
and
groEL
protocols
confirmed
the
presence
of
R.
monacensis
in
I.
boliviensis
tick
from
Costa
Rica.
Most
ticks
were
infected
with
a
single
pathogen,
however,
four
R.
sanguineus
s.l.
ticks
infected
with
E.
canis
displayed
also
infections
with
A.
platys
(2),
A.
phagocytophilum
CR2
(1)
and
R.
amblyommii
(1),
whereas
one
R.
sanguineus
s.l.
tick
showed
mixed
infection
of
A.
phagocytophilumCR1
and
A.
platys-like,
and
what
is
extensively
reported
in
the
literature
(Latrofa
et
al.,
2014).
E.
chaffeensis
and
E.
ewingii
were
not
detected
in
the
present
study.
They
have
been
reported
in
dog
ticks
in
the
United
States
using
molecular
techniques
(Breitschwerdt
et
al.,
1998;
Varde
et
al.,
1998),
and
are
primarily
transmitted
by
Amblyomma
americanum.
This
tick
is
widely
distributed
in
the
southeastern
United
States
and
northern
of
New
York
(Means
and
White,
1997),
but
has
not
been
reported
in
Costa
Rica
(Álvarez
et
al.,
2005).
Mainly
R.
sanguineus
s.l.
ticks
were
analyzed
in
the
present
study,
what
could
explain
why
E.
chaffeensis
and
E.
ewingii
were
not
detected.
The
presence
of
these
agents
in
ticks
of
other
regions
should
not
be
ruled
out,
and
it
is
recommended
to
continue
investigations
to
detect
these
agents
in
Costa
Rica.
5.
Conclusions
This
study
reports
the
identification
of
tick-borne
pathogens
in
dog
ticks
of
Costa
Rica,
some
of
them
of
zoonotic
interest,
point-
ing
out
the
increasing
awareness
of
diseases
associated
to
these
ectoparasites,
and
suggesting
a
risk
for
the
emergence
of
tick-borne
diseases
in
dogs
and
humans
of
Costa
Rica.
Further
studies
are
needed
to
investigate
the
presence
of
Rickettsiales
in
the
country,
to
establish
the
epidemiological
and
clinical
importance
of
these
pathogens,
and
to
set
up
surveillance
plans
for
zoonotic
diseases
transmitted
by
ticks.
Acknowledgments
This
work
was
supported
by
Fondo
Especial
para
el
Finan-
ciamiento
de
la
Educación
Superior,
Consejo
Nacional
de
Rectores
(FEES-CONARE)
and
Vicerrectoría
de
Investigación,
Universidad
Nacional
(UNA),
Costa
Rica.
Leyda
Ábrego
was
research
fellow
from
the
Deutscher
Akademischer
Austauschdienst
(DAAD)
during
this
investigation.
We
wish
to
thank
the
Center
of
Disease
Control,
Atlanta,
USA,
for
donating
DNA
from
HL-60
infected
cells
with
A.
phagocytophilum
(strain
Trestom),
and
Roman
Ganta,
from
the
Kansas
State
University,
USA,
for
submitting
plasmids
containing
segments
of
DNA
from
E.
canis,
E.
chaffeensis
and
E.
ewingii.
Thanks
to
Jorge
Zavala,
Universidad
Autonoma
de
Yucatan,
Mexico,
for
pro-
viding
Rickettsia
felis
DNA
that
was
used
as
positive
control.
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