Steve. Sviat for preparing the culture media and. Larry. Kirk for performing the FA tests. LITERATURE. CITED .... G. H. ADLER,. AND. A. SPIELMAN. 1990. Short- ...
Journal
EXPERIMENTAL (TAMIAS
INFECTION
STRIATUS)
SPIROCHETE Robert 1
WITH
THE
(BORRELIA
G. McLean,
Division
OF THE
Sonya
of
EASTERN
LYME
Wildlife
29(4),
Diseases,
1993,
pp.
527-532
CHIPMUNK
DISEASE
BURGDORFERI)
R. Ubico,2
and Lynita
M. Cooksey3
Diseases, National Center for Infectious Diseases, P.O. Box 2087, Fort Collins, Colorado 80522, USA
of Vector-Borne
Infectious
Centers for Disease Control, 2 Department of Fishery and Wildlife
Biology,
Collins, Colorado 80523, USA Department of Environmental Health, Fort Collins, Colorado 80523, USA
Colorado
State University,
Fort
Lyme
ABSTRACT:
northeast,
a
and
striatus)
may
is found
as an
serve
host,
11
isolated
adult
tected
by
lasted
2 to mo,
All
spirochetes 5 days.
and
Ixodes
106
were
isolated
became
infected
burgdorferi,
with Lyme
reservoir
disease,
caused
disease,
and Wisconsin and now cases
ported in 46 states, indigenous in far a!.,
1985;
the
spirochete
for
1991). than
Only 13 states 100 indigenous
eight
of those
and the Wisconsin, souri
fredisinifoci,
states
Disease
had reports cases during in the
states were Michigan
myscus
leucopus) in
United 1984;
States Loken
1985;
Donahue
and
the and
deer
enzootic midwestern
B.
1987;
all
of
animals,
after
de-
chipmunks,
and
at
1 wk
and
Laboratory-reared
feeding
experimental
Westchester
starting
post-inoculation.
as
were
eight
on
two
infection,
of
the
inoculated
Tamias
chipmunks,
mouse species also are major immature stages of the pri-
mary
tick
merly
I. dammini).
vector,
Ixodes
most
Control,
eastern second
chipmunk to mice
of more 1990;
with B. mature
is et
were
the
in
chipmunk
that reported more than of Lyme disease in 1990
mice
(P. manof the of the
is found
a!.,
et a!., 527
deer
mice
such
as residential
in
important
striatus) was of infection
in
12 of
the
species could and reservoir of, white-footed some
settings yards
13 states cases This
become an in addition mice or and
locations
in the
northeast
in
ences
patterns.
movement
of imeastern
100 human (Hall, 1981).
or woods and yards western states, because dance and differences and
small
Massachusetts and in Wishowever, the
and as a host ticks. The
Georgia, and Mis-
small mammal important host to, or in place
deer
is the major host this tick. White-
(Tamias prevalence
burgdorferi I. scapularis
(for-
white-tailed
reservoir in coastal (Mather et al., 1989) (Godsey et a!., 1987);
northeast
regions states
scapularis
The
mice
burgdorferi mice (Pero-
Godsey
These for the
mamma! (USA) consin
(Anderson and Magnarelli, et a!., 1985; Levine et et a!.,
of
eight
coast,
potential burgdorferi
of
footed
(USA).
The principal hosts for are thought to be white-footed iculatus) northeastern
ears
in
Pacific
Spirochetemias
of
(USA), during have been re-
were
other five California,
area
infected.
(Odocoileus virginianus) for the adult stages
although the disease fewer states (Schmid
Centers
the
strains
the
chipmunk
their of B.
a hyperendemic
days
1987). hosts by
along
in
eastern
competence.
Borrelia burgdorferi, is now the most quently reported vector-borne human ease in the United States. The disease tially was recognized in two small Connecticut the 1970’s
the 133
spirochetes
INTRODUCTION
Lyme
in
media at
except from
became from
organs
The
To characterize
spirochetes
scapularis
is endemic
States.
regions
locations.
chipmunks
internal
burgdorferi,
United
Barbour-Stoenner-Kelly
various
larval I. scapularis ticks chipmunks. Key words: Borrelia St na tus, Ixodes scapularis,
the
in some with
and
in
of
disease-enzootic
host
inoculated
Spirochetes
from
states
inoculated
leucopus
Borrelia
spirochete
the
reservoir were
(USA).
isolating
for 4
York
the coastal
throughout
chipmunks
New
State University,
by
Pacific
important
Peromyscus
from
County,
for
caused
north-central,
(Tanilas and
disease,
Colorado
the upper midof its local abunin habitat prefer-
JOURNAL
528
Our tentia! ervoir
OF WILDLIFE
DISEASES,
VOL. 29, NO. 4, OCTOBER
objective was to evaluate of the eastern chipmunk host for B. burgdorferi.
MATERIALS
Immature
the poas a res-
AND METHODS
eastern
chipmunks
were
captured
in 1988 in Madison, Wisconsin, during a time when Madison was not known to be endemic for either I. scapularis ticks or indigenous human cases of Lyme disease (J. J. Kazmierczak, pers. comm.), and they were shipped to the Cen-
ters Fort
for Disease Control (CDC) laboratory in Collins, Colorado (USA). They were maintained at the CDC laboratory for about 18 mo prior
the
to
munks
of the experiment. The chiphoused individually in clear plastic wire tops and were provided with start
were
cages
with
food
and
ad libitum
water
periment except ticks. Chipmunks
oxyflurane Mundelein,
when were
and with hydrochloride
ketamine
Laboratories,
Inc.,
Fort
placement.
Briefly,
was
as
Eleven
follows. experimental
Dodge, our
Iowa,
chipmunks
inoculated
were
ilar
fashion
the
control
to the animals
the
culture
media
housed
eight
and
in
and
two
sep-
three were
animals)
and three control handled in a sim-
experimental
was
for
design I with
(Group
chipmunks
USA)
experimental
groups
Group II with with spirochetes
and
animals
Inc.,
of (25 to 30 mg/kg) of (Vetalar, Fort Dodge
5 mg
tick
ex-
exposed to with meth-
(Metofane, Pitman-Moore, Illinois, USA) for the collection
specimens
arate
the
throughout
they were anesthetized
ones.
sham
One
inoculated
sampled
on
of
with
the
same
schedule as the experimental animals in Group II and the other two controls were euthanized with carbon dioxide, and necropsied. The blood, ear, liver, spleen, kidney and bladder were tested after the termination of the experiment. All animals except for two controls were tested for current infection with spirochetes prior to their use in the experiment. During the tick feeding phase of 6 days, two chipmunks from Group II (animal numbers 59 and 72) were individually held in wire cages suspended over water to collect detached, replete ticks. Approximately 50 1. scapularis larvae from a laboratory colony maintained at CDC
were placed on each anethesized oculated with spirochetes 4 mo engorged
placed
larvae
in glass
collected
vials
from
covered
chipmunk previously.
the with
water fine with
inThe were nylon 70 to
mesh and held in a desiccator jar 80% relative humidity and maintained at about 24 C and 10 hr light per day. The ticks were allowed to molt to the nymphal stage before they were tested for spirochete infection. Twenty unfed larvae from the same batch of ticks
1993
that had been applied to the tested for infection as well.
The isolates the chipmunks bour-Stoenner-Kelly
of spirochetes were originally
chipmunks
were
used to inoculate isolated in Barculture medium I. scapularis ticks
(BSK) (Barbour, 1984) from adult collected within the Lyme disease hyperendemic region in Westchester County, New York (USA) (41#{176}8’N,73#{176}48’W)during November 1989, and from ear biopsy tissue from a white-footed mouse (NY9O-14) captured in Westchester County, New York in June 1990 (McLean, unpub!.). The isolates tentatively were identified by the direct fluorescent antibody (FA) test using a polyclonal antibody against B. burgdorferi produced locally in New Zealand white rabbits
(Lord et a!., 1992). The isolates were confirmed as B. burgdorferi by the indirect fluorescent antibody test using anti-OpsA monoclonal antibody H5322 (Barbour et al., 1983) with known strains of B. burgdorferi (B31) and B. hermsii as positive and negative controls, respectively. The isolates were passed in BSK medium and inocula of the second passage level containing about 106 spirochetes/mi of BSK medium were prepared. Three chipmunks in Group I each were inoculated subcutaneously (sc) with 0.1 ml of the tick isolate to determine the infectiousness of cultured spirochetes inoculated by needle and
syringe. Following the conclusion of the preliminary experiment, eight chipmunks in experimental Group II each were inoculated subcutaneously with 0.1 ml or about 10 spirochetes of isolate
NY9O-14.
Blood specimens for culturing were collected weekly for the
of spirochetes 4 wk from
first
the suborbital sinus with capillary pipets from chipmunks in Group I and daily for the first 8 days from chipmunks in Group II. Several drops of whole blood from each animal were dispensed directly into 7 ml of BSK medium for spirochete isolation. Skin biopsies from the ears were collected weekly for 8 wk. To collect ear biopsy
tized
specimens,
chipmunks
the ear surfaces of anesthewere cleansed thoroughly with
70% ethyl alcohol (ETOH) before a small portion (3 to 4 mm in diameter) of ear tissue was punched out from each animal (Sinsky and Piesman, 1989). The tissue specimens were dipped first into 5% hydrogen peroxide solution and then 70% ETOH and washed with sterile phosphate buffered saline (PBS) before being placed into BSK medium for spirochete isolation.
Upon
completion
of the
experiment,
chip-
munks were euthanized with carbon dioxide gas, and a blood sample was taken from the heart. Necropsies were performed on the eight chipmunks from Group II only, and the three control animals. During necropsy, small portions (about 1 cm2) of the liver, spleen, kidney, bladder and
McLEAN
Isolation of spirochetes in BSK culture with Borrelia burgdorfeni.
TABLE 1. inoculated
ET AL.-LYME
media
from
tissues
DISEASE
INFECTiON
of eastern
chipmunks
Blood
0
Control
2
3
4
5
6
7
8
0
group
+
-
-
-
-
2
3
4
5
6
7
8
+
-
+
+
+
+
+
-
+
+
-
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
-
-
-
-
+
+
+
+
+
-
-
+
5
8
7
3
0
0
0
0
7
6
7
6
7
7
7
7
+
+
+
+
58
-
-
+
+
+
59
-
+
-
+
+
61
-
+
-
+
67
-
+
+
+
68
-
-
+
+
71
-
-
+
72
-
-
0
4
Numberspositive negative
for isolation,
+
spirochetes
=
+
isolated
aseptically
before
and
in BSK
as and pestle in enough BSK medium to make approximately 10% suspensions. Nymphal ticks that had fed upon the two chipmunks from Group II were pooled in groups of five, their surface cleaned with hydrogen peroxide, alcohol, and PBS, and ground in BSK media with a mortar and pestle. The unfed larvae were pooled in two groups of 10 and treated similarly. The suspensions were centrifuged at 1,200 RPM for 10 mm and 0.2 ml of each supernatant fluid was inoculated into 7 ml of BSK medium. All cultures were maintained at 34 C and examined weekly for 8 wk or until spirochetes were observed by dark-field microscopy. The spirochetes isolated in culture from the ticks feeding on the chipmunks were confirmed by the FA test. The data were analyzed with a chi-square test (Sokal and Rohlf, 1981). were
above,
removed
with
maceration
a
spirochetes
cleansed
rochete
were
blood
experiment
(Table
inoculated became
with infected
isolated
or ear tissue prior to the 1).
A!!
different
weekly blood samples chipmunks in Group post-inoculation (P1)
at
Group
II
strain isolation
in
were
first
from
2
3.4
detected to
± 0.3
had
on
5 days days
tissue
itive
for
ble
1).
tissue and
the at
three 1 wk for spi-
all
1). on
all
eight
A!! day
3 P1.
The
Group
from
from
the
remaining
animal
generally
remained
tissues
all
eight isolates
Larval
readily
in removing
taching
from
completed the engorged a! stage. ture and of
14
positive P1, not on
attached
7
day
wk
were
chipmunks
ear day
by 8
occasionally
ticks
by
for
and head of the two though one chipmunk cessful
animals
chipmunks
ear
II (Ta-
isolated
eight
of
culture-pos-
in
were
lasted
(±SE)
were
of
Ear
and
animals
seven
P1.
by
eight
in
for
two
1 P1
animals
Spirochetes
NY9O-14
detected
a mean
specimens
1).
taken from I starting were negative
day
(Table
some
Four
of
Spirochetemias
with
a spirochetemia
punch
the as
media.
from
0.003).
tissues
with
culture
(Table
spirochetes control an-
spirochetes
ear
a spirochetemia
weeks
room and from free of infection
