Article

Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores

Division of Medical Microbiology, Department of Pathology, Johns Hopkins School of Medicine, 21205 Baltimore, MD, USA.
Ticks and Tick-borne Diseases (Impact Factor: 2.72). 06/2012; 3(4):247-53. DOI: 10.1016/j.ttbdis.2012.04.002
Source: PubMed

ABSTRACT

The present study aims to detect and characterize by molecular techniques, the presence of tick-borne pathogens in wild captive carnivore blood samples from Brazil. Blood was collected from 76 Brazilian felids, 23 exotic felids, 3 European wolves (Canis lupus), and 97 Brazilian canids maintained in captivity in zoos located in São Paulo and Mato Grosso states, Brazil. DNA of each sample was used in PCR reactions for Ehrlichia, Anaplasma, and Rickettsia identification. The blood from 10/100 (10%) of canids (1 European wolf, 3 bush dogs, and 6 crab-eating foxes) and from 21/99 (21%) felids (4 pumas, 6 little spotted cats, 4 ocelots, 3 jaguarundis, 1 tiger, and 3 lions) contained fragments of 16S rRNA gene of Ehrlichia spp. Fragments of Anaplasma spp. groESL and 16S rRNA genes were detected in the blood of 1/100 (1%) canids (1 bush dog) and in 4/99 (3%) felids (4 little spotted cats), respectively. Rickettsia species infections were not identified. The present work showed that new strains of Ehrlichia and Anaplasma spp. circulate among wild carnivores in Brazil.

Full-text

Available from: John Stephen Dumler, Jul 14, 2015
Ticks
and
Tick-borne
Diseases
3 (2012) 247–
253
Contents
lists
available
at
SciVerse
ScienceDirect
Ticks
and
Tick-borne
Diseases
journa
l
h
o
mepage:
www.elsevier.de/ttbdis
Short
communication
Molecular
detection
of
tick-borne
bacterial
agents
in
Brazilian
and
exotic
captive
carnivores
M.R.
André
a,b
,
J.S.
Dumler
b
,
D.G.
Scorpio
b
,
R.H.F.
Teixeira
c
,
S.M.
Allegretti
d
,
R.Z.
Machado
a,
a
Departamento
de
Patologia
Veterinária,
Faculdade
de
Ciências
Agrárias
e
Veterinárias
(FCAV),
Universidade
Estadual
Paulista
(UNESP),
14884-900
Jaboticabal,
SP,
Brazil
b
Division
of
Medical
Microbiology,
Department
of
Pathology,
Johns
Hopkins
School
of
Medicine,
21205
Baltimore,
MD,
USA
c
Zoológico
de
Sorocaba,
18020-268
Sorocaba,
SP,
Brazil
d
Departamento
de
Parasitologia,
Universidade
Estadual
de
Campinas
(Unicamp),
13084-971
Campinas,
SP,
Brazil
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
10
October
2011
Received
in
revised
form
19
April
2012
Accepted
24
April
2012
Keywords:
Ehrlichia
Anaplasma
Rickettsia
Wild
carnivores
Brazil
a
b
s
t
r
a
c
t
The
present
study
aims
to
detect
and
characterize
by
molecular
techniques,
the
presence
of
tick-borne
pathogens
in
wild
captive
carnivore
blood
samples
from
Brazil.
Blood
was
collected
from
76
Brazilian
felids,
23
exotic
felids,
3
European
wolves
(Canis
lupus),
and
97
Brazilian
canids
maintained
in
captivity
in
zoos
located
in
São
Paulo
and
Mato
Grosso
states,
Brazil.
DNA
of
each
sample
was
used
in
PCR
reactions
for
Ehrlichia,
Anaplasma,
and
Rickettsia
identification.
The
blood
from
10/100
(10%)
of
canids
(1
European
wolf,
3
bush
dogs,
and
6
crab-eating
foxes)
and
from
21/99
(21%)
felids
(4
pumas,
6
little
spotted
cats,
4
ocelots,
3
jaguarundis,
1
tiger,
and
3
lions)
contained
fragments
of
16S
rRNA
gene
of
Ehrlichia
spp.
Fragments
of
Anaplasma
spp.
groESL
and
16S
rRNA
genes
were
detected
in
the
blood
of
1/100
(1%)
canids
(1
bush
dog)
and
in
4/99
(3%)
felids
(4
little
spotted
cats),
respectively.
Rickettsia
species
infections
were
not
identified.
The
present
work
showed
that
new
strains
of
Ehrlichia
and
Anaplasma
spp.
circulate
among
wild
carnivores
in
Brazil.
© 2012 Elsevier GmbH. All rights reserved.
Introduction
Arthropod-borne
diseases
are
of
major
global
importance
to
human
and
animal
health.
The
epidemiology
of
these
diseases
involves
several
infectious
agents,
hosts,
and
vectors
(Harrus
and
Baneth,
2005).
Both
arthropods
and
arthropod-transmitted
infec-
tions
are
expanding
their
zoogeographic
range
due
to
climatic,
ecological,
and
environmental
changes.
The
presence
of
domestic
animals
in
wildlife
environments
has
resulted
in
an
increased
asso-
ciation
between
wildlife
reservoirs
and
vector
species
with
human
and
pet
activities
(Shaw
et
al.,
2001).
Although
globally
important,
human
tick-borne
diseases
remain
poorly
studied
in
Brazil,
where
confirmed
human
Brazilian
spotted
fever
cases
have
been
reported
in
13
states
(De
del
Fiol
et
al.,
2010).
In
Minas
Gerais
state,
anti-
bodies
to
E.
chaffeensis
were
identified
among
healthy
humans
and
in
patients
with
clinical
signs
compatible
with
tick-borne
diseases
(Calic
et
al.,
2004;
Costa
et
al.,
2005,
2006).
Corresponding
author
at:
Laboratório
de
Imunoparasitologia,
Departamento
de
Patologia
Veterinária,
Faculdade
de
Ciências
Agrárias
e
Veterinárias
Júlio
de
Mesquita
Filho
(UNESP),
Campus
de
Jaboticabal,
Via
de
Acesso
Prof.
Paulo
Donato
Castellane,
s/n,
Zona
Rural,
CEP:
14884-900,
Jaboticabal,
São
Paulo,
Brazil.
Tel.:
+55
16
3203
2663;
fax:
+55
16
3202
4275.
E-mail
address:
zacarias@fcav.unesp.br
(R.Z.
Machado).
While
the
occurrence
of
tick-borne
agents
in
domestic
animals
in
Brazil,
mainly
in
dogs,
has
been
extensively
evaluated,
there
are
only
few
reports
concerning
the
identification
of
tick-borne
agents
in
Brazilian
free-ranging
and
captive
wild
carnivores.
The
molec-
ular
detection
of
E.
chaffeensis
in
Brazilian
marsh
deer
(Blastocerus
dichotomus)
(Machado
et
al.,
2006)
raised
the
importance
of
investi-
gating
wildlife
reservoirs
for
tick-borne
zoonotic
agents.
Antibodies
to
Ehrlichia
spp.
have
been
detected
in
free-ranging
(Filoni
et
al.,
2006)
and
captive
neotropical
felids
(André
et
al.,
2010;
Filoni
et
al.,
2012).
In
addition,
Ehrlichia
spp.
DNA
has
been
detected
in
5
Brazilian
wild
felid
species
(André
et
al.,
2010;
Widmer
et
al.,
2011).
In
a
previous
molecular
survey,
we
found
11
(15%)
out
of
72
neotropical
captive
wild
felids
contained
Ehrlichia
spp.
DNA
in
blood
(André
et
al.,
2010).
Six
of
the
7
existing
species
of
Brazil-
ian
wild
felids,
including
ocelots
(Leopardus
pardalis),
little
spotted
cats
(Leopardus
tigrinus),
margays
(Leopardus
wiedii),
pampas
cats
(Oncifelis
colocolo),
jaguars
(Panthera
onca),
and
pumas
(Puma
con-
color),
and
2
of
the
4
existing
species
of
Brazilian
wild
canids,
namely
bush
dogs
(Speothos
venaticus)
and
maned
wolves
(Chryso-
cyon
brachyurus)
are
endangered
(www.ibama.gov.br).
Thus,
we
aimed
to
detect
and
characterize,
using
molecular
techniques,
the
presence
of
tick-borne
pathogens
in
additional
neotropical
and
exotic
wild
felids
and
canids
maintained
in
captivity
in
various
locations
throughout
Brazil.
1877-959X/$
see
front
matter ©
2012 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.ttbdis.2012.04.002
Page 1
248 M.R.
André
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 247–
253
Table
1
Number,
species,
and
zoo
location
of
sampled
wild
canids
and
felids.
Species
Common
name
Location
(N)
Total
Felis
colocolo
Pampas
cat
So
(1)
1
Leopardus
pardalis Ocelot Am
(2),
IS
(2),
So
(1),
Bau
(3),
Ita
(2),
Mg
(1),
Pir
(4)
15
Leopardus
tigrinus Little
spotted
cat
Am
(1),
IS
(2),
So
(14),
SC
(1)
Ita
(1),
Pir
(3),
Cat
(1),
NO
(2)
25
Leopardus
wiedii
Margay
Am
(2)
2
Panthera
onca
Jaguar
AMC
(1),
Am
(1),
Bau
(1),
SP
(3)
6
Puma
concolor
Puma
Rp
(1),
Am
(2),
So
(3),
Cat
(2)
8
Puma
yagouaroundi
Jaguarondi
Am
(2),
IS
(4),
So
(4),
SC
(2),
Bau
(3),
Ita
(3),
Pir
(1)
19
Panthera
tigris
a
Tiger So
(2),
Ita
(2),
SP
(4) 8
Caracal
caracal
a
Caracal SP
(1)
1
Leptailurus
serval
a
Serval
SP
(1)
1
Panthera
leo
a
Lion
Rp
(1),
Am
(1),
So
(2),
Bau
(3),
SP
(5)
12
Prionailurus
viverrinus
a
Fishing
cat
SP
(1)
1
Speothos
venaticus
Bush
dog
Am
(8),
IS
(1),
Pi
(6),
SP
(8),
NO
(1),
Cui
(3)
27
Cerdocyon
thous Crab-eating
fox Rp
(9),
Am
(3),
Ara
(6),
IS
(3),
So
(2),
SC
(4),
Bau
(1),
Ita
(1),
Pir
(3),
Cat
(6),
Le
(1) 39
Cerdocyon
brachyurus
Maned
wolf
Rp
(2),
Am
(1),
Ara
(1),
IS
(2),
So
(2),
Ita
(3),
Mg
(3),
SP
(3),
Cat
(1),
NO
(3),
Bau
(2)
23
Pseudalopex
vetulus Hoary
fox
Rp
(1),
Am
(2),
Ara
(3),
IS
(1),
So
(1),
8
Canis
lupus
a
European
wolf
SP
(3)
3
AMC,
Association
Mata
Ciliar,
Jundiaí,
São
Paulo;
SC,
São
Carlos
Zoo,
São
Paulo;
NO,
Nova
Odessa
Zoo,
São
Paulo;
Ped,
Pedreira
Zoo,
São
Paulo;
Le,
Leme
Zoo,
São
Paulo;
Cps,
Campinas
Zoo,
São
Paulo;
Bau,
Bauru
Zoo,
São
Paulo;
Cui,
Cuiabá
Zoo,
Mato
Grosso;
Cat,
Catanduva
Zoo,
São
Paulo;
Rp,
Ribeirão
Preto,
São
Paulo;
Ita,
Itatiba
Zoo,
São
Paulo;
Am,
Americana
Zoo,
São
Paulo;
Mg,
Mogi
Mirim
Zoo,
São
Paulo;
Ara,
Arac¸
atuba
Zoo,
São
Paulo;
Pir,
Piracicaba
Zoo,
São
Paulo;
IS,
Ilha
Solteira
Zoo,
São
Paulo;
SP,
São
Paulo,
São
Paulo.
a
Exotic
carnivores.
Materials
and
methods
Five
mL
of
blood
was
collected
from
each
of
76
Brazilian
wild
captive
felids,
23
exotic
captive
felids,
3
captive
European
wolves
(Canis
lupus),
and
97
Brazilian
wild
canids
maintained
in
zoos
located
in
São
Paulo
and
Mato
Grosso
states
(Table
1).
All
samples
were
collected
under
the
Brazilian
Institute
of
Envi-
ronment
and
Renewable
Natural
Resources
license
numbers
#S02027.002943/2005
and
#15901-1.
Animals
were
immobilized
with
a
mixture
of
ketamine
(Francotar
®
,
Virbac,
Carros
Cedex,
France)
(10
mg/kg)
and
xylazine
(Francotar
®
,
Virbac,
Carros
Cedex,
France)
(1
mg/kg).
Physical
examination
of
each
animal
did
not
reveal
any
clinical
signs
of
disease,
and
no
ticks
were
found
on
any
of
the
sampled
animals.
DNA
was
extracted
from
200
L
of
whole
blood
using
the
QIAamp
DNA
Blood
Mini
kit
(QIAGEN,
Valencia,
CA,
USA)
according
to
the
manufacturer’s
instructions.
Each
sample
of
DNA
was
used
as
template
in
conventional
PCR
assays
with
genus-
and
species-specific
rrs
(16S
rRNA
gene)
primers
for
Ehrlichia
canis
(Murphy
et
al.,
1998),
E.
chaffeensis
(Kocan
et
al.,
2000),
E.
ewingii
(Persing
et
al.,
1996),
Anaplasma
spp.
(Massung
et
al.,
1998),
and
A.
platys
(Inokuma
et
al.,
2001b).
PCR
assays
for
Ehrlichia/Anaplasma
spp.
based
on
dsb
(Doyle
et
al.,
2005),
groESL
(Sumner
et
al.,
1997;
Lotric-Furlan
et
al.,
1997;
Nicholson
et
al.,
1999),
ftsz
(Lee
et
al.,
2003),
rpoB
(Taillardt-Bisch
et
al.,
2003),
omp-1
(Inayoshi
et
al.,
2004),
and
gltA
(Inokuma
et
al.,
2001a)
genes
were
performed
for
additional
molecular
characterization.
Ehrlichia
spp.
(E.
canis,
E.
chaffeensis)
and
Anaplasma
spp.
(A.
phago-
cytophilum,
A.
platys)
DNA-positive
controls
were
included
in
all
PCR
assays.
Negative
domestic
cat
and
dog
blood
samples
and
ultra-pure
water
(Promega)
were
used
as
negative
and
no-template
controls,
respectively.
The
PCR
amplifications
were
performed
in
a
Gradient
Cycler
(Perkin-Elmer
TM
model
PT-200).
Specific
real-
time
5
nuclease
PCR
reactions
for
A.
phagocytophilum
msp-2
and
E.
chaffeensis
vlpt
(Reller
et
al.,
2009)
were
performed
on
samples
con-
taining
16S
RNA
genes
of
Ehrlichia/Anaplasma
spp.
in
conventional
PCR
assays.
Similarly,
a
5
nuclease
real-time
PCR
was
performed
for
spotted
fever
(ompA)
and
typhus
group
Rickettsia
(17-kDa
lipopro-
tein
gene)
as
previously
described
(Prakash
et
al.,
2009).
These
assays
were
conducted
using
a
BioRad
CFX384
real-time
PCR
ana-
lyzer
and
1
L
blood
DNA
in
duplicate.
The
sensitivities
of
these
real-time
assays
based
upon
amplification
of
dilutions
of
amplicons
cloned
into
plasmids
were
1–10
copies/L
blood
for
A.
phagocy-
tophilum,
E.
chaffeensis,
and
Rickettsia
spp.
For
positive
PCRs,
purified
amplicons
(QIAquick
Gel
Extraction
kit,
QIAGEN,
Valencia,
CA,
USA)
were
ligated
into
the
pGEM-T
Easy
vector
(Promega)
and
transformed
into
competent
JM109
Escherichia
coli
(Promega;
Madison,
Wisconsin,
USA).
Clones
were
selected
by
blue/white
colony
screening;
plasmid
DNA
was
isolated
using
QIAprep
Miniprep
Kit
(QIAGEN,
Valencia,
CA,
USA)
and
submitted
for
sequence
determination
(ABI
Prism
310
Genetic
Analyser
Applied
Biosystem/Perkin
Elmer
[Foster
City,
CA,
USA]).
Consensus
DNA
sequences
were
obtained
through
the
analysis
of
sense
and
antisense
sequences
using
the
CAP3
program
(http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py).
Com-
parisons
with
sequences
deposited
in
GenBank
were
performed
using
BLAST
(Altschul
et
al.,
1990).
BioEdit,
CLUSTAL
X
(Thompson
et
al.,
1994),
and
TreeView
programs
were
used
for
alignment
and
phylogenetic
analysis,
respectively.
The
neighbor-joining
distance
method
was
used
to
build
the
phylogenetic
tree
(Saitou
and
Nei,
1987).
Bootstrap
analysis
with
1000
replications
was
used
to
estimate
the
confidence
of
branching
patterns
of
the
trees
(Felsenstein,
1985).
Results
The
blood
of
10/100
(10%)
canids
(1
European
wolf,
3
bush
dogs,
and
6
crab-eating
foxes)
and
21/99
(21%)
felids
(4
pumas,
6
little
spotted
cats,
4
ocelots,
3
jaguarundis,
1
tiger,
and
3
lions)
contained
fragments
of
16S
rRNA
gene
of
Ehrlichia
spp.
Fifteen
wild
carnivores
(11
felids
and
4
canids)
contained
ehrlichial
DNA
closely
related
to
E.
chaffeensis
by
BLAST
analysis
(Table
2).
In
addition,
16
of
the
wild
carnivores
(10
felids
and
6
canids)
also
contained
ehrlichial
DNA
closely
related
to
E.
canis
by
BLAST
analysis
(Table
2).
With
reduced
confidence
(bootstrap
value
of
348/1000
itera-
tions),
the
phylogenetic
analysis
of
the
amplified
350-bp
16S
rRNA
gene
fragment
positioned
the
ehrlichial
DNA
in
a
clade
including
E.
canis
and
E.
chaffeensis
(Fig.
1).
However,
no
samples
contained
E.
chaffeensis
vlpt
genes
when
subjected
to
specific
real-time
PCR.
Anaplasma
spp.
DNA
was
detected
in
the
blood
of
1/100
(1%)
canids
(1
bush
dog)
and
in
4/99
(4%)
felids
(4
little
spotted
cats).
Based
on
16S
rRNA
gene
PCR
amplification,
the
Anaplasma
sp.
found
in
wild
felids
was
closely
related
to
A.
phagocytophilum
by
BLAST
analysis
(Table
2).
Using
groESL
PCR
amplification,
the
Anaplasma
sp.
found
in
one
wild
canid
was
closely
related
to
A.
phagocy-
tophilum
by
BLAST
analysis
(Table
2).
Page 2
M.R.
André
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 247–
253 249
Table
2
Results
of
molecular
survey
for
Ehrlichia
spp.
and
Anaplasma
spp.
in
captive
wild
carnivores
in
Brazil.
Strain
ID
Host
species
Locality
Target
gene
GenBank
accession
number
Closest
GenBank
entry
similarity
(by
BLAST)
%
similarity
CH
LT
IS1
Little
spotted
cat
Ilha
Solteira
16S
rRNA
JQ260838
Ehrlichia
chaffeensis
98%
CH
OI
S1
Ocelot
Sorocaba
16S
rRNA
JQ260839
Ehrlichia
chaffeensis
98%
CH
PC
S1
Puma
Sorocaba
16S
rRNA
JQ260840
Ehrlichia
chaffeensis
98%
CH
PT
S1
Tiger
Sorocaba
16S
rRNA
JQ260841
Ehrlichia
chaffeensis
98%
CH
LT
S2
Little
spotted
cat
Sorocaba
16S
rRNA
JQ260842
Ehrlichia
chaffeensis
98%
CH
LT
S3
Little
spotted
cat
Sorocaba
16S
rRNA
JQ260843
Ehrlichia
chaffeensis
98%
CH
PC S1 Puma Sorocaba 16S
rRNA JQ260857
Ehrlichia
chaffeensis
98%
CH
PT
S2
Tiger
Sorocaba
16S
rRNA
JQ260844
Ehrlichia
chaffeensis
98%
CH
PY
SC1
Jaguarundi
São
Carlos
16S
rRNA
JQ260845
Ehrlichia
chaffeensis
98%
CH
PL
B1
Lion
Bauru
16S
rRNA
JQ260846
Ehrlichia
chaffeensis
98%
CH
LP
P1
Ocelot
Piracicaba
16S
rRNA
JQ260847
Ehrlichia
chaffeensis
98%
CH
CL
SP1
European
wolf
São
Paulo
16S
rRNA
JQ260858
Ehrlichia
chaffeensis
98%
CH
CT
AM1 Crab-eating
fox Americana 16S
rRNA JQ260846 Ehrlichia
chaffeensis
– 98%
CH
CT
CAT1
Crab-eating
fox
Catanduva
16S
rRNA
JQ260859
Ehrlichia
chaffeensis
98%
CH
CT
CAT2
Crab-eating
fox
Catanduva
16S
rRNA
JQ260859
Ehrlichia
chaffeensis
98%
EC
PC
CAT1
Puma
Catanduva
16S
rRNA
JQ260848
Ehrlichia
canis
98%
EC
LT
NO1 Little
spotted
cat Nova
Odessa 16S
rRNA JQ260849
Ehrlichia
canis
98%
EC
LT
NO2
Little
spotted
cat
Nova
Odessa
16S
rRNA
JQ260850
Ehrlichia
canis
99%
EC
PC
AM1
Puma
Americana
16S
rRNA
JQ260851
Ehrlichia
canis
98%
EC
PL
AM1
Lion
Americana
16S
rRNA
JQ260849
Ehrlichia
canis
98%
CH
PY
B1
Jaguarundi
Bauru
16S
rRNA
JQ260852
Ehrlichia
canis
98%
EC
PL
B2 Lion Bauru 16S
rRNA JQ260853 Ehrlichia
canis
98%
EC
LP
I1
Ocelot
Itatiba
16S
rRNA
JQ260854
Ehrlichia
canis
99%
EC
PY I1 Jaguarundi Itatiba
16S
rRNA
JQ260855
Ehrlichia
canis
98%
EC
LP
P2
Ocelot
Piracicaba
16S
rRNA
JQ260856
Ehrlichia
canis
98%
EC
SV
CUI1
Bush
dog
Cuiabá
16S
rRNA
JQ260860
Ehrlichia
canis
98%
EC
SV
CUI2
Bush
dog
Cuiabá
16S
rRNA
JQ260860
Ehrlichia
canis
98%
EC
CT
ARA1
Crab-eating
fox
Arac¸
atuba
16S
rRNA
JQ260860
Ehrlichia
canis
98%
EC
CT
RP1 Crab-eating
fox Ribeirão
Preto 16S
rRNA JQ260861
Ehrlichia
canis
99%
EC
SV
P1
Bush
dog
Piracicaba
16S
rRNA
JQ260861
Ehrlichia
canis
99%
EC
CT
P1
a
Crab-eating
fox
Piracicaba
16S
rRNA
JQ260860
Ehrlichia
canis
98%
A
LT
S1
Little
spotted
cat
Sorocaba
16S
rRNA
JQ260835
Anaplasma
phagocytophilum
98%
A
LT
S2
Little
spotted
cat
Sorocaba
16S
rRNA
JQ260837
Anaplasma
phagocytophilum
97%
A
LT
S3
Little
spotted
cat
Sorocaba
16S
rRNA
JQ260836
Anaplasma
phagocytophilum
99%
A
LT
S4
Little
spotted
cat
Sorocaba
16S
rRNA
JQ260837
Anaplasma
phagocytophilum
97%
EC
CT P1
a
Crab-eating
fox Piracicaba
dsb
JQ248011
Ehrlichia
canis
99%
A
SV
P1
Bush
dog
Piracicaba
groESL
JQ248010
Anaplasma
phagocytophilum
97%
a
Same
strain.
Identical
sequences
are
represented
by
a
single
accession
number.
The
fragments
of
Anaplasma
spp.
16S
rRNA
gene
found
in
wild
felids
were
in
a
clade
with
A.
phagocytophilum
and
A.
platys.
Although
A.
platys
could
be
reasonably
distinguished
from
A.
phagocytophilum
(bootstrap
values
628/1000),
the
Anaplasma
spp.
detected
in
wild
felids
more
consistently
grouped
with
A.
phagocy-
tophilum
(bootstrap
values
994/1000)
(Fig.
1).
Phylogenetic
analysis
based
on
groESL
(1297
bp)
demonstrated
that
the
Anaplasma
spp.
found
in
a
bush
dog
was
in
a
clade
with
A.
phagocytophilum
and
A.
bovis.
Moreover,
a
high
bootstrap
value
(962/1000)
separated
A.
phagocytophilum
from
A.
bovis
and
the
Anaplasma
spp.
found
in
a
bush
dog
(Fig.
2).
However,
all
samples
with
Anaplasma
spp.
16S
rRNA
and
groESL
genes
detected
in
nested
PCR
assays
did
not
contain
A.
phagocytophilum
msp2
in
specific
real-time
PCRs.
Samples
were
also
negative
in
PCR
assays
broadly
targeting
Anaplasmataceae
rpoB,
ftsZ,
gltA,
and
omp-1
genes.
Rickettsia
species
DNA
was
not
identified
in
any
of
the
blood
samples
tested.
Discussion
Carnivores
play
a
role
as
sentinels
for
vector-borne
pathogens
because
they
can
act
as
hosts
for
both
bacteria
and
arthropod
vectors,
and
because
they
have
more
widely
distributed
activity
than
tick
hosts
(Foley
et
al.,
1999).
The
identification
of
vector-
borne
agents
infecting
wild
carnivores
could
help
to
establish
vector-borne
disease
risk
areas
for
humans
and
animals.
Also,
with
regard
to
wild
carnivore
conservation,
monitoring
these
infec-
tions
is
important
for
the
management
of
endangered
populations
(Daszak
et
al.,
2000).
Morulae
resembling
Ehrlichia
sp.
in
monocytes
and
mononuclear
cells
have
been
identified
in
a
lioness
exhibit-
ing
clinical
signs
compatible
with
feline
ehrlichiosis
(Buoro
et
al.,
1994).
Wild
dogs
(Lycaon
pictus)
experimentally
infected
with
E.
canis
demonstrated
clinical
signs
of
disease
(Van
Heerden,
1979).
This
study
describes
the
frequency
of
Anaplasmataceae-infected
captive
wild
carnivores
in
Brazil,
as
detected
by
molecular
tech-
niques.
Although
serological
studies
demonstrate
antibodies
to
these
agents
in
wild
canids
around
the
world
(Alexander
et
al.,
1994;
Waner
et
al.,
1999;
Pusterla
et
al.,
1999,
2000;
Shamir
et
al.,
2001;
Groen
et
al.,
2002)
and
felids
(Ryser-Degiorgis
et
al.,
2005;
Filoni
et
al.,
2006;
André
et
al.,
2010),
few
studies
have
attempted
a
comprehensive
molecular
characterization
of
diverse
Anaplasmataceae
agents
in
wild
carnivores.
Our
work
detected
ehrlichial
DNA
in
a
high
proportion
of
wild
felids
and
canids.
In
a
preliminary
study,
we
detected
ehrlichial
DNA
in
wild
felids
from
São
Paulo
state
and
Distrito
Federal,
Brazil
(André
et
al.,
2010).
Despite
the
similarity
of
the
16S
rRNA
gene
sequences
detected
in
wild
felids
apparently
infected
with
E.
canis,
a
phylogenetic
analysis
based
on
omp-1
DNA
sequences
demon-
strated
that
the
Ehrlichia
sp.
detected
in
these
captive
felids
is
likely
positioned
between
Ehrlichia
and
Anaplasma
species,
suggesting
a
new
species
or
strain
parasitizing
wild
felids
(André
et
al.,
2010).
Although
31
wild
captive
felids
and
canids
had
ehrlichial
DNA
in
blood
by
16S
rRNA
gene
nested
PCR,
none
contained
ehrlichial
DNA
when
tested
by
PCR
based
on
omp-1
or
other
genes
(gltA,
rpoB,
ftsZ,
and
groESL),
limiting
additional
phylogenetic
inferences.
The
vari-
able
successful
amplification
of
different
genes
could
be
explained
by
the
very
low
level
of
bacteremia
in
blood
samples.
Moreover,
Page 3
250 M.R.
André
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 247–
253
0.01
Rickettsia rickettsii (DQ150691)
Neorickettsia helminthoeca (U12457)
Neorickettsia sennetsu (CP000237)
Neorickettsia risticii (CP001431)
994
884
Anaplasma platys (EF139459)
Anaplasma sp. A_LT_S4
Anaplasma phagocytophilum (AY055469)
Anaplasma sp. A_LT_S1
975
994
628
Anaplasma bovis (U03775)
Anaplasma ovis
(AF414870)
Anaplasma centrale (AF414869)
Anaplasma marginale (M60313)
993
813
550
490
Neoehrlichia lotoris (EF633744)
Neoehrlichia mikurensis
(GU227699 )
639
Ehrlichia muris
(AB196302)
Ehrlichia sp. from Ixodes ovatus ticks (AB028319)
416
415
Ehrlichia sp. EC_SV_CUI2
Ehrlichia sp. EC_LT_NO1
Ehrlichia sp. EC_CT_ARA1
Ehrlichia sp. EC_CT_P1
Ehrlichia canis (CP000107)
Ehrlichia sp. EC_PC_CAT1
495
487
483
481
528
Ehrlichia ruminantium (CR767821)
Ehrlichia sp CH_LT_S1
Ehrlichia sp. CH_CT_CAT2
Ehrlichia sp. CH_PC_S1
Ehrlichia sp. CH_EW_SP1
Ehrlichia ewingii (M73227)
Ehrlichia sp. CH_PT_S1
Ehrlichia sp. CH_CT_CAT1
31
6
Ehrlichia chaffeensis (CP000236)
1
12
89
204
221
288
348
828
819
Fig.
1.
Phylogenetic
positions
of
the
Anaplasmataceae
detected
in
captive
carnivores
in
Brazil
based
on
16S
rRNA
sequences
(350
bp).
The
tree
was
constructed
using
the
neighbor-joining
method,
and
numbers
on
the
tree
indicate
bootstrap
values
for
branch
points.
Accession
numbers
are
in
parentheses.
The
inset
shows
the
number
of
nucleotide
substitutions
per
1000
bp.
PCR
protocols
aimed
at
amplification
of
different
genes
performed
in
our
study
may
have
proved
unsuitable
for
amplification
of
vari-
ants
of
Anaplasma
and
Ehrlichia
species
that
infect
wild
carnivores
in
Brazil.
The
phylogenetic
analysis
based
on
a
short
16S
rRNA
gene
fragment
(350
bp)
did
not
provide
sufficient
genetic
discrimination
to
identify
the
Ehrlichia
species
in
carnivores,
despite
additional
unsuccessful
attempts
to
amplify
larger
fragments.
Based
on
phy-
logenetic
analysis
of
16S
rRNA
gene
and
dsb
partial
sequences,
an
Ehrlichia
sp.
found
in
2
jaguars
from
the
Pantanal,
Mato
Grosso
state,
Brazil,
grouped
into
a
cluster,
albeit
distantly,
with
differ-
ent
genotypes
of
E.
ruminantium
(Widmer
et
al.,
2011),
suggesting
the
existence
of
more
than
one
strain
or
new
species
of
Ehrlichia
circulating
among
Brazilian
wild
carnivores.
E.
canis
DNA
was
not
detected
in
blood
samples
of
97
wild
or
51
exotic
felids
maintained
in
captivity
in
the
São
Paulo
Zoologi-
cal
Park,
when
a
specific
real-time
PCR
was
used,
which
suggests
that
infections
with
this
pathogen
among
felids
are
not
problematic
in
captive
settings
(Filoni
et
al.,
2012).
In
our
study,
the
presence
of
E.
canis
infecting
wild
felids
cannot
be
ruled
out,
since
a
specific
real-time
PCR
for
this
agent
was
not
performed.
These
data
demon-
strate
the
first
molecular
detection
of
Ehrlichia
and
Anaplasma
spp.
in
captive
canids
in
Brazil.
Here,
E.
canis
infection
was
confirmed
in
a
crab-eating
fox.
In
some
parts
of
Brazil,
including
the
geographic
area
where
samples
were
collected,
canine
monocytic
ehrlichiosis
caused
by
E.
canis
is
a
significant
tick-borne
disease
(Nakaghi
et
al.,
2008;
Dagnone
et
al.,
2009).
In
Brazil,
E.
chaffeensis
infects
marsh
deer
(Blastocerus
dichotomus),
which
could
act
as
a
natural
reser-
voir
(Machado
et
al.,
2006).
Among
wild
carnivores,
E.
chaffeensis
has
been
detected
in
coyotes
from
Oklahoma
in
the
USA
(Kocan
et
al.,
2000).
However,
E.
chaffeensis
infection
in
the
Brazilian
wild
carnivores
was
ruled
out
by
specific
real-time
PCR
targeting
the
E.
chaffeensis
vlpt
gene.
Anaplasma
spp.
DNA
was
identified
only
in
4
felids
and
in
one
canid.
Although
many
of
the
PCR
assays
targeting
other
genes
were
negative,
this
could
be
explained
by
very
low
level
bacteremias
yielding
bacterial
DNA
quantities
in
blood
samples
below
the
detection
limit
of
the
PCR
protocols
used.
It
is
also
possible
that
the
primers
utilized
for
amplification
of
genes
characterized
for
existing
species
might
not
be
suitable
for
amplification
of
variants
of
Ehrlichia
and
Anaplasma
circulating
in
Brazilian
wild
carnivores.
Successful
amplification
of
A.
phagocytophilum
based
on
ankA,
groESL,
and
the
intergenic
23S-5S
rRNA
region
varied
considerably
Page 4
M.R.
André
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 247–
253 251
0.1
Rickettsia prowazekii (Y15783)
Neorickettsia helminthoeca (AY050315)
Neorickettsia risticii (U96732 )
Neorickettsia sennetsu (U88092)
1000
1000
Anaplasma platys (AY008300 )
Anaplasma centrale (HM057223)
Anaplasma phagocytophilum (AF172163)
Anaplasma sp. A_SV_P1
Anaplasma bovis
748
962
1000
Anaplasma marginale (AF414860 )
Anaplasma ovis (FJ460440 )
1000
459
1000
Candidatus Neoehrlichia mikurensis (EU810407)
Candidatus Neoehrlichia lotoris (EF633745 )
1000
Ehrlichia ruminantium (U13638)
Ehrlichia ewingii (AF195273)
Ehrlichia chaeensis (L10917)
Ehrlichia muris
(AF210459)
Ehrlichi a sp from Ixodes ovatus ticks (AB032711 )
999
835
Ehrlichia canis (U96731)
994
996
1000
985
1000
Fig.
2.
Phylogenetic
positions
of
the
Anaplasmataceae
detected
in
captive
carnivores
based
on
the
groESL
gene
sequences
(1297
bp).
The
tree
was
constructed
using
the
neighbor-joining
method,
and
numbers
on
the
tree
indicate
bootstrap
values
for
branch
points.
Accession
numbers
are
in
parentheses.
The
inset
shows
the
number
of
nucleotide
substitutions
per
1000
bp.
among
diverse
wild
species
in
the
western
USA
(Rejmanek
et
al.,
2012).
In
Brazil,
A.
phagocytophilum
DNA
was
recently
detected
in
dog
blood
samples
from
Rio
de
Janeiro
state
(Santos
et
al.,
2011).
Regarding
the
occurrence
of
Anaplasma
sp.
infection
in
wild
canids,
A.
phagocytophilum
DNA
has
been
detected
in
pumas
(Foley
et
al.,
1999)
and
coyotes
(Pusterla
et
al.,
2000)
from
California,
USA.
Recently,
A.
phagocytophilum
DNA
was
not
detected
among
148
captive
wild
felid
blood
samples,
demonstrating
a
low
prevalence
among
wild
felids
in
captive
settings
(Filoni
et
al.,
2012).
In
phy-
logenetic
analyses
based
on
16S
rRNA
gene
and
groESL
sequences,
Anaplasma
spp.
found
in
wild
felids
and
canids
clustered
in
the
same
clade
as
A.
phagocytophilum
and
A.
bovis.
Thus,
the
Anaplasma
spp.
found
in
these
wild
carnivores
could
represent
a
new
strain
or
species.
The
ecological
and
epidemiological
significance
of
genetic
and
phenotypic
variants
of
Anaplasma
spp.
is
still
poorly
understood
(Rejmanek
et
al.,
2012).
All
animals
were
negative
for
Rickettsia
species
DNA,
which
could
be
explained
by
the
low
number
of
Rickettsia
bacteria
circulat-
ing
in
blood
and
the
limited
period
of
rickettsemia
(Dantas-Torres,
2007).
Rickettsiosis
is
a
re-emerging
problem
in
Brazil,
where
capy-
baras
and
opossums
are
the
most
probable
amplifying
hosts
for
R.
rickettsii,
as
identified
from
Amblyomma
cajennense
and
A.
aureo-
latum
ticks
found
on
small
rodents
(Labruna,
2009).
Antibodies
to
spotted
fever
group
(SFG)
rickettsiae
have
been
detected
in
rodents
and
opossums
in
both
non-endemic
(Pacheco
et
al.,
2007)
and
endemic
(Pena
et
al.,
2009)
areas
for
Brazilian
Spotted
Fever
(BSF).
Also,
antibodies
to
Rickettsia
species,
namely
R.
rickettsii,
R.
park-
eri,
R.
amblyommii,
R.
rhipicephali,
R.
felis,
and
R.
bellii,
have
been
recently
detected
in
10
free-ranging
jaguars
in
Brazil,
although
no
rickettsial
DNA
was
found
in
the
blood
of
these
felids
(Widmer
et
al.,
2011),
which
is
not
surprising.
Most
recently,
R.
amblyommii
have
been
detected
in
Amblyomma
longirostre
ticks
parasitizing
wild
birds
in
the
Atlantic
Forest
in
northeastern
Brazil
(Ogrzewalska
et
al.,
2011).
Although
we
believe
that
these
pathogens
are
likely
transmit-
ted
by
ticks,
we
found
no
observable
ticks
parasitizing
the
animals
during
examination.
The
use
of
acaricides
for
vector
control
is
a
common
practice
in
Brazilian
zoos.
Also,
there
is
limited
confir-
mation
regarding
how
long
these
animals
have
been
maintained
in
captivity,
which
makes
ecological
interpretation
of
vector
bur-
den
and
transmission
challenging,
whether
infection
occurred
in
captivity
or
in
the
natural
environment.
In
conclusion,
molecular
characterization
reveals
a
variable
infection
rate
caused
by
Ehrlichia
and
Anaplasma
spp.
among
Brazilian
wild
carnivores,
which
are
closely
related
to
E.
canis,
E.
chaffeensis,
and
A.
phagocytophilum.
Future
studies
should
be
Page 5
252 M.R.
André
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 247–
253
conducted
which
aim
to
detect
and
amplify,
with
higher
sensitiv-
ity,
longer
DNA
sequences
to
improve
phylogenetic
comparisons.
Moreover,
efforts
to
isolate
detected
agents
will
allow
for
improved
genetic
and
antigenic
characterization.
Despite
additional
molecu-
lar
characterization,
the
present
work
highlights
the
possibility
of
new
strain
or
species
emergence
of
Ehrlichia
and
Anaplasma
spp.
among
wild
Brazilian
carnivores,
and
whether
unique
phenotypic
manifestations
of
disease
exists
will
be
the
target
of
future
inves-
tigations.
The
role
which
wild
carnivores
may
play
in
the
ecology
and
epidemiology
of
vector-borne
diseases
in
Brazil
still
needs
to
be
determined.
Conflict
of
interest
None
declared.
Acknowledgments
The
authors
would
like
to
thank
Fundac¸
ão
de
Amparo
à
Pesquisa
do
Estado
de
São
Paulo
(FAPESP)
for
the
scholarship
(number
#07/59889-6)
and
financial
support
(number
#08/55570-8),
and
CAPES
(Coordenac¸
ão
de
Aperfeic¸
oamento
de
Pessoal
de
Nível
Supe-
rior)
for
sandwich-scholarship
(#1483-10-2);
Instituto
Brasileiro
do
Meio
Ambiente
e
dos
Recursos
Naturais
Renováveis
(IBAMA)
for
the
concession
of
licences
(numbers
#S02027.002943/2005
and
#15901-1)
for
collecting
and
packaging
blood
samples
from
wild
canids
and
felids;
and
Zoológico
de
Brasília,
Zoológico
Municipal
Bosque
dos
Jequitibás
de
Campinas,
Zoológico
de
Pedreira,
Bosque
Municipal
Fábio
Barreto
de
Ribeirão
Preto,
Zoológico
de
Ameri-
cana,
Zoológico
de
Arac¸
atuba,
Zoológico
de
Sorocaba,
Zoológico
de
Leme,
Zoológico
de
São
Carlos,
Zoológico
de
Itatiba,
Zoológico
de
Mogi
Mirim,
Zoológico
de
Piracicaba,
Zoológico
de
Nova
Odessa,
Zoológico
de
Americana,
Zoológico
de
Catanduva,
Zoológico
de
Cuiabá,
and
Fundac¸
ão
Parque
Zoológico
de
São
Paulo.
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    • "Although the presence of E. canis infection is likely to occur in both Indian communities (due to the presence of the vector R. sanguineus), it is possible that other ehrlichial agents, associated with native tick species, also occur in the region and therefore could be causing serological cross-reactions with E. canis antigens. In fact, recent studies have reported a variety of ehrlichial agents different from E. canis infecting ticks and animals in different regions of Brazil, indicating that the diversity of Ehrlichia spp. in Brazil is much greater than previously thought (Machado et al. 2007, Widmer et al. 2011, André et al. 2012, Cruz et al. 2012, Machado et al. 2012, Almeida et al. 2013, Aguiar et al. 2014). "
    [Show abstract] [Hide abstract] ABSTRACT: With the aim of studying some tick-borne diseases, a total of 327 dogs (114 from Tapirapé and 213 from Karajá indigenous ethnicity, Mato Grosso, MT, Brazil) were sampled. Serum samples were submitted to the indirect fluorescence antibody test (IFAT) to detect antibodies against Babesia vogeli, Ehrlichia canis, and Rickettsia spp. Possible associations of risk factors and the occurrence of seroreativity to tick-borne agents and tick infestations were analyzed through chi-squared tests. Among 327 dogs, 46 (13.15%) were seropositive for B. vogeli and 47 (14.37%) for E. canis. The B. vogeli seroprevalence was higher for Karajá and for adult dogs (p>0.05). No association was found for E. canis seroprevalence. From 103 serum samples tested with rickettsial antigens, 90 (87%) dogs were seropositive to Rickettsia spp., with highest reactivity to Rickettsia amblyommii. Canine seropositivity to Rickettsia spp. was associated (p<0.05) with ethnicity (higher seroprevalence in Tapirapé dogs), age (higher in adults), and hunting (higher among hunting dogs). From the 327 dogs, 39 (11.9%) were infested by ticks (Amblyomma cajennense sensu stricto, Amblyomma ovale, Amblyomma oblongoguttatum, Amblyomma tigrinum, and Rhipicephalus sanguineus). Infestations by Amblyomma spp. ticks were higher in dogs from Tapirapé community and in hunting dogs (p<0.05). Regarding R. sanguineus, infestations were higher (p<0.05) among young dogs.
    Full-text · Article · Jul 2015 · Vector borne and zoonotic diseases (Larchmont, N.Y.)
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    • "From Asia, it was detected in Turkey, Russia, China, Korea and Japan (Aktas et al., 2011; Cao et al., 2000; Ohashi et al., 2005; Kim et al., 2006). Reports of A. phagocytophilum or closely related strains from Africa and South America are occasional (Inokuma et al., 2005; André et al., 2012 André et al., , 2014). There is no reliable data available about its occurrence in South America and Australia. "
    [Show abstract] [Hide abstract] ABSTRACT: Granulocytic anaplasmosis is a common vector-borne disease of humans and animals with natural transmission cycle that involves tick vectors, among which Ixodes ricinus is the most important. The present paper reports the prevalence and geographical distribution of A. phagocytophilum in 10,438 questing Ixodes ricinus ticks collected at 113 locations from 40 counties of Romania. The unfed ticks were examined for the presence of A. phagocytophilum by PCR targeting a portion of ankA gene. The overall prevalence of infection was 3.42%, with local prevalences ranging between 0.29% and 22.45%, with an average prevalence of 5.39% in the infected localities. The infection with A. phagocytophilum was detected in 72 out of 113 localities and in 34 out of 40 counties. The highest prevalence was recorded in females followed by males and nymphs. The results and the distribution model have shown a large distribution of A. phagocytophilum, covering Romania's entire territory. This study is the first large scale survey of the presence of A. phagocytophilum in questing I. ricinus ticks from Romania. Copyright © 2015 Elsevier GmbH. All rights reserved.
    Full-text · Article · Mar 2015 · Ticks and Tick-borne Diseases
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    • "These genera are recognized vectors of pathogenic bacteria with medical and veterinary relevance in neotropical regions [3]. In South America, information about the occurrence of tick-borne bacteria in wild mammals, which are frequently exposed to tick-bites, is limited [4,5]. Moreover, several severe and economically important diseases of livestock in tropical regions are caused by tick-borne pathogens (i.e. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Ixodid ticks play an important role in the transmission and ecology of infectious diseases. Information about the circulation of tick-borne bacteria in ticks is lacking in Ecuador. Our aims were to investigate the tick species that parasitize Andean tapirs and cattle, and those present in the vegetation from the buffer zone of the Antisana Ecological Reserve and Cayambe-Coca National Park (Ecuador), and to investigate the presence of tick-borne bacteria.Methods Tick species were identified based on morphologic and genetic criteria. Detection of tick-borne bacteria belonging to Rickettsia, Anaplasma, Ehrlichia and Borrelia genera was performed by PCRs.ResultsOur ticks included 91 Amblyomma multipunctum, 4 Amblyomma spp., 60 Rhipicephalus microplus, 5 Ixodes spp. and 1 Ixodes boliviensis. A potential Candidatus Rickettsia species closest to Rickettsia monacensis and Rickettsia tamurae (designated Rickettsia sp. 12G1) was detected in 3 R. microplus (3/57, 5.3%). In addition, Anaplasma spp., assigned at least to Anaplasma phagocytophilum (or closely related genotypes) and Anaplasma marginale, were found in 2 A. multipunctum (2/87, 2.3%) and 13 R. microplus (13/57, 22.8%).Conclusions This is the first description of Rickettsia sp. in ticks from Ecuador, and the analyses of sequences suggest the presence of a potential novel Rickettsia species. Ecuadorian ticks from Andear tapirs, cattle and vegetation belonging to Amblyomma and Rhipicephalus genera were infected with Anaplasmataceae. Ehrlichia spp. and Borrelia burgdorferi sensu lato were not found in any ticks.
    Full-text · Article · Jan 2015 · Parasites & Vectors
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