(If miiliuf
Jmirual
ISOLATION
OF BARTONELLA
SPP.
NEONATES
OF NATURALLY
INFECTED
Michael Y. Kosoy,1 Russell and James E. Chllds2
Olga
L. Regnery,
Division of Viral and Rickettsial Diseases, Prevention, Atlanta, Georgia, 30333, USA 2 Corresponding author (e-mail:
[email protected])
National
FROM
EMBRYOS
1995. pp
AND
RODENTS
I. Kosaya,
Center
l42
Diwa.ses.
Dana
for Infectious
C. Jones,
Diseases,
Eric
Centers
L Marston
for Disease
Control
and
Embryos and neonatal offspring of wild-captured cotton rats (Sigrnodon hispidus) white-footed mice (Peromyscus leucopus) were tested for the presence of Bartonella Spp. Isolates of Bartonella spp. were obtained from 18 of 31 embryos and 7 of 19 neonates from bacteremic dams of the two species; no isolates were obtained from material from non-bacteremic dams. Sequence analysis demonstrated that the isolates from embryos and neonates matched the phylogenetic group of Bartonella spp. isolates obtained from the mother. No antibodies to homologous Bartonella spp. antigens were detected in maternal and neonatal blood or embryonic tissue. These findings suggest the possibility of vertical transmission of Bartonella spp. among natural rodent hosts. Key words: Bartonella spp., embryos, immune tolerance, infection transmission, Perornyscus leucopus. rodents, Sigrnodon hispidus, zoonotic disease. ABSTRACT:
and
INTRODUCTION
The
prevalence spp.
tonella
Several
species
of the
genus including
Rochalimaea),
(formerly
Bartonella B.
rodent
1994;
with
Bar-
communities
>50% (Birtles Kosoy et at., 1997).
typically hen-
of bacteremia
within
is
and Harrison, Bacteremic and
selae,
B.
and B. elizabethae, cause human disease in the United States. Human infection can lead to a wide spectrum of illnesses, ranging from cat-scratch
non-bacteremic animals either lack, or have very low titers of, antibodies to these organisms (Kosoy et at., 1997). The mech-
disease 1996).
to bacillazy The number
nella
spp.
been
suggested
quintana,
the
genus
the and
last several characterization
nella from
(formerly wild-captured
ed
Harrison,
Our tenance nella vectors B.
and the 1994;
human Cat
infected
fleas
States et at.,
spp. and
is;
of
Byam
(Birtles mainBarto-
the
and
B.
henselae
83#{176}72’W), (30#{176}62’N,
of
State
cats to humans
and
(Tyzzer,
Barto-
fleas
have
1942).
genus
Lloyd, may
(Zangwill
et
Amelia 81#{176}46’W),
Island, and
vertical transmisBartonella spp. embryonal, and rodents. Rolive traps (H. B. Florida, USA) USA (33#{176}66’N, Florida, USA
Morrow
Mountain
Park,
retroorbital
plexus.
Ten
be
at., et
1993). 305
hispidus)
and
pregnant four
pregnant
cotton
rats
whitefooted mice (Peromyscus leucopus) were captured and deeply anesthetized, and 49 fettuses or uteri containing small embryos were removed and placed into sterile vials. In some instances only a single embryo was removed for testing, although the pregnant female may have (Sigrnodon
of Bar-
(Chomel
AND METHODS
North Carolina, USA (35#{176}36’N, 80#{176}14’W), from May 1996 through May 1997. Captured animals were anesthetized with methoxyflurane (Metofane, PittmanMoore, Mundelein, Illinois, USA) and bled from the
can cycle
acquire
although
To assess the potential for sion, we attempted to isolate simultaneously from maternal, neonatal tissues of wild-caught dents were captured by using Sherman Traps, Tallahassee, from Social Circle, Georgia,
quintana can be human body louse
transmission
among possibly
B.
rodents
unclear,
MATERIALS
1997).
(Ctenocephalidesfelis)
with in the
tonella 1996)
United Kosoy
which
are
over
spp. isolated from the Unit-
(sandflies while by the
by
with the description of numerous Barto-
Grahamella) rodents
bacillzformis
important
at.,
increased
anisms
in
spp. is still rudimentary. Arthropod play a role in the transmission
(Pediculus be
years
(Koehler, included
understanding of the natural and transmission cycle of
Lutzomyia), transmitted 1920).
has
Bartonella
Kingdom
and
angiomatosis of species
JOURNAL
306
had
additional
tuses
were
OF WILDLIFE
DISEASES,
embryos.
In
separated
within a biosafety phosphate-buffered moved from their technique. Estimated
tamed
by measuring
1968).
Fotur
the
from
34, NO. 2, APRIL
laboratory,
placental
hood, rinsed saline (pH amniotic sacs gestational
lengths
S. hispidus
VOL.
with sterile 7.4), and reusing aseptic age was ob-
of fetuses
gave
fe-
tisstues
birth
(Rugh,
in the
traps.
These 19 newborn rodents were anesthetized, bled by cardiac puncture, and measured. Blood, placental and embryonic tissue samples were stored at -70 C until they were used. Details
of the procedures tused to isolate spp. have been published previously (Regnery et al., 1992a). Whole or diluted blood and 10% suspensions of embryonal tissue in brain heart infusion medium (Becton Dickinson, Cockeysvihle, Maryland, USA) were used for isolation. Aliquots of 0.1 ml of the blood or tissue stuspensions were applied to heart infusion agar plates supplemented with 5% rabbit
1998
Antigen
wells
suspensions,
BALB/c of
were
overlaid
or mouse
mice
2,048.
with
of
conjugate
ratories
Inc.,
serum,
fluid
homologotus
Mixtures
hamster
with
ascitic
tissue
produced
in
reciprocal
anti-mouse
titers
and
(Kirkegaard
& Perry
Gaithersburg,
Maryland,
anti-
LaboUSA)
beled with fluorescein isothiocyanate as appropriate. The same conjugates
were
ha-
used
have given good results when used against a variety of sera from diverse rodent species, including S. hispidus and P leucopus, in other studies (Kosoy et al., 1996). Sera and tissue suspension were screened at a dilution of 1:32.
Bartonella
blood
(Becton
Dickinson).
The
plates
were
in-
cubated at 32 C in an aerobic atmosphere of 5% CO2 and held for 5 to 15 days. The cultiures were examined daily for bacterial growth and colonies tentatively identified as Bartonella spp. were streaked onto a new agar plate. When visually uncontaminated Bartonella-like colony counts were >500 per plate, material was collected, placed in brain heart infusion medium,
and
stored
at
Isolated belonging pared for
using
-70
C.
Tissue
Kit
(Qiagen,
Inc.,
Chat-
sworth, California). Primers homologous to the citrate synthase (gltA) gene of B. henselae Hotuston I were used, as described previously (Norman et al., 1995), to amplify a 379-bp
product. directly.
The PCR products were sequenced Sequences were analyzed by STADEN (Staden, 1982) and Wisconsin Sequence Analysis Package 8.1 Unix (Genetic Computer Group, Inc., Madison, Wisconsin, USA) and aligned with B. henselae. B. quintana. B. yinsonii, groups (Kosoy
and
four newly described phylogenetic C, and D) of the genus Bartonella et al., 1997). The gltA gene sequences have GenBank accession numbers of L38987, B. henselae; U28073, B. quintana; U28074, B. vinsonil; U84372, group A; U84375, group B; U84377, group C; and U84379, group D. (A,
The
B,
indirect
performed al., 1992b;
fluorescence
as previously Kosoy et al.,
antibody
spot slides were dotted with antigens selae (ATCC 49882), B. quintana 358), lates and
B.
vinsonii
(ATCC
test
was
described (Regnery et 1997). Wells of 12-well
VR-152),
of (ATCC
and
representing phylogenetic groups D) of Bartonella obtained from
B.
henVR-
four iso(A, B, C rodents.
spp.
Bartonella
of four pregnant from six of 10 colony
counts
mary from
plating 1,000
were
of bacteria of to
agar
to
and
embryos)
or
placental tissues nine bacteremic
not
of
any rats
(Table spp.
Bartonella
bryos
after
pri-
obtained
sethe P
phylogenetic group from S. hispidus begroup A (five of six
1). Bartonella
(Table
from
and The
Citrate synthase gene demonstrated that isolates from pregnant
isolated from from eight of cotton
three
mice rats.
blood varied colony-forming
400,000
belonged D, while the isolates longed to phylogenetic females
from
on
maternal
leucopus
five adults)
isolated
white-footed pregnant cotton
units (CFU)/ml. quence analysis three Bartonella
adult
organisms tentatively identified as to the genus Bartonella were prepolymerase chain reaction (PCR) by
QIAamp
RESULTS
the
four
C (one of spp. were obtained dams, but
nonbacteremic
1). were isolated from emfrom each of the bactere-
mic females of P leucopus bryos recovered from four teremic S. hispidus (Table
and from emof the five bac1). The colony
counts plating varied analysis
of bacteria on agar after primary of the 10% embryonal suspensions from 4 to 200 CFU/ml. Sequence demonstrated that the three Bar-
tonella
isolates
from
to phylogenetic cultured from longed No
to phylogenetic
isolates
were
and
cotton
rat which
tonella
phylogenetic
emic
removed cotton
rats.
while
was
belonged 15 isolates embryos be-
group
obtained
the placenta
tissues
embryos
P leucopus
group D, S. hispidus
A (Table
from
removed
from
bacteremic group from
C, four
1).
embryonic with or
the Bar-
from
nonbacter-
14
KOSOY
TABLE
Bartone!la
1.
/mispidus)
in
spp.
obtained
frouii
ET AL.-BARTONELLA
emiibrvos
Ceorgia
of
amid
IN EMBRYOS
bacteremic
North
white-footed
Carolina,
AND
mice
NEONATES
leucopus)
(P.
OF RODENTS
and
307
cotton
rats
(S.
1996-1997. Phvlogemmetic group of Bartonella (numnher ol isolates scjtuemuced) frommm:
Emnbrvos Nmimmi-
Species
Locality
P leucopus
Morrow North
P leucopus
Morrow North
P. leucopus
Morrow Northi
S. /mi.spidus
Social
amid date
Momuntaimi
State
Caroliusa,
May
her in litter
I .emugth (mmumuu)
Pmmsitive/ tested
Staternal
3
12
1/1
D
(1)
D
(1)
1
20
1/1
1)
(1)
D
(I
2
17
1/1
1)
(1)
D
Park.
Placental tissue
blood
Emnhrommal tissime 1)
(1)
I)
(I)
(1)
1)
(1)
(4)
1996
Momumutain
State
Carolina,
May
Park,
)
1996
Momuuitaiui
State
Carolina,
May
Park, 1996
Circle,
Ceorgia,
Atugust
4
11
4/4
A (I)
A
(1)
A
Circle,
Ceorgia,
August
6
40
0/6
A (1)
A
(1)
uiouie
Circle.
Ceorgia.
March
3
3/3
A
A
(3)
A (3)
Circle,
Ceorgia,
April
5
40
0/5
C (1)
none
uione
Circle,
Ceorgia,
May
6
42
3/5
A
(I)
A
(1)
A (3)
Circle.
Ceorgia,
May
8
12
5/5
A
(1)
A
(1)
A
1996 S. /ii.spidu.s
Social 1996
S. Imispidus
Social
7
(1)
1997 S.
lzispidus
Social 1997
S. /zi.spidu.s
Social
1997 S. /zi.spk/us
Social
(5)
1997 Pluvlogemuetic isolates
group
of phvlogenetic
The
was determnimued h the similarity of the gItA gemme to seqtuemmces groups A. B. C, and D (Kosov et al.. 1997).
gestation
period
genus 1968),
Peromyscus
bryos
of P leucopus
days
(Meyer
and
the
in
averages
lengths
rodents
of
of the
the
with
infected
em-
pidus
of
Meyer,
infected
neonates
to
age. cotton
2.
days
rats
inside
1944),
in
of
