Wildlife. Diseases,. 27(2),. 1991, pp. 248-253. U) Wildlife. Disease. Association. 1991. SEROEPIDEMIOLOGY. OF LEPTOSPIROSIS. IN MINNESOTA. WOLVES.
Journal
SEROEPIDEMIOLOGY Muhammad
A. Khan,
Department
of Veterinary
University
of Minnesota,
(Mailing
address:
Sagar
North
Central
collected
Forest
tumnalls,
ballum, and
of L. grippotyphosa, penhageni. No indicate that L. Key words: serovars
miology,
zoonosis,
1992
=
1986
the
most
world
and
et at.,
infected vironment McCulloch,
1989).
become infected urine or indirectly contaminated
1970;
moist
stow-moving can survive
by
the
(Diesch hosts contact with soil,
and can
horses;
and headache, failure
pathy,
1981;
tospirosis
1983a).
interrogans
Adolf
acute
is
febrite
Fame,
Weit
first
1986).
The
disease
in Germany
with
pigs; in my-
occasional and Tn-
In humans,
reported
in
(Khan
wild
species
can
act
interrogans
USA Minnesota
and
for
tep-
1886
other
(Odocoileous (Ingebrigtsen
virginianus) et a!., 1986).
of the
study
present
55108,
USA)
wild
or
do-
was
of Minnesota The objective to determine
the
of 12 selected serovars of in wolves (Canis lupus) (USA).
MATERIALS
by
Diesch,
hageni,
1987).
Many
St. Paul,
AND
METHODS
Four hundred fifty seven serum samples were collected from wolves in northern Minnesota that were live-trapped and anesthetized. Blood was collected in glass tubes by saphenous or cephalic venipuncture. Serum was separated by centrifugation and stored at -20 C until tested. Most samples were collected during June through October 1972 to 1986 and were tested in 1987. These sera have been used in previous surveys for canine pathogens (Mech et al., 1986; Goyat et a!., 1986). The following L. Interrogans serovars were included in the study: australls, autumnalla, ballum, batavlae, bratislava, canicola, copen-
characterized
and (Hanson
L.
stagnant
illness
meningitis in humans
was
20708,
Maryland
Avenue,
seroprevalence L. interrogans from Minnesota
with urine-
by abortion and stillbirth in cattle and periodic ophthalmia (moon blindness) a!gia, kidney
en-
streams where L. for weeks (Roth,
et a!.,
L.
Laurel, FoIwell
reported in foxes ( Vulpes sp.) (Clark et a!., 1961), coyotes (Canis latrans) (Cirone et a!., 1978; Drewek et a!., 1981), and jackals (Canis sp.) (van der Hoeden, 1955). Serological evidence of !eptospirosis has recently been reported in white-tailed deer
of the
into
directly by by contact
Hathaway
a
interro-
kidneys
their urine Susceptible
water,
ponds and interrogans caused
Leptospira
is excreted
through 1966).
by
the genus LepAlt pathogenic
in the
and
zoo-
under one species, contains more than into 19 serogroups
occurs
host
H. Fritts2
mestic animals and even for humans (Hathaway et al., 1981; Hathaway and Blackmore, 1981). Leptospirosis has been
mammals is caused
of 1984).
leptospires are classified L. interrogans, which 170 serovars organized usually
Steven
from and were test. Twelve
distributed
affects
pathogenic spirochete tospira (Thiermann,
(Nielsen
and
Medicine,
457)
of
is a widely
which
gans
Mech,2
WOLVES
survey.
Leptospirosis throughout
L David
of Veterinary Center,
Station,
INTRODUCTION
nosis
IN MINNESOTA
L Diesch,
College
Experiment
(n
samples
from 1972 through using a microtiter agglutination
pyrogenes,
Stanley
Investigation,
27(2), 1991, pp. 248-253 Disease Association 1991
Diseases, U) Wildlife
wolves (Canis lupus) in northern Minnesota were tested for antibodies against Leptospira interrogans serovars included in the study were: australls, aubataviae, bratislava, canicola, copenhageni, grippotyphosa, hard Jo, pomona, tarassovi. Fifty-two (11%) sera had antibody titers of 1:50 against one or more Interrogans. The seroprevatence of different serovars in decreasing order was: bratislava, autumnalis, canicola, pomona, ballum, pyrogenes, hard Jo, and coantibodies were found against australia, batavlae, and tarassovi. These results interrogans infection may occur in wolves of Minnesota. Leptospirosis, Leptospira interrogans, serovars, wolves, Canis lupus, seroepide-
Serum
ABSTRACT:
M. Goyal,1
Diagnostic
St. Paul, Minnesota 55108, USA Service, Patuxent Wildlife Research
Fish and Wildlife
2
OF LEPTOSPIROSIS
Wildlife
of
grippotyphosa, tarassovi.
genes, and incriminated
as reservoirs 248
hardJo,
pomona,
These serovars as a cause of disease
pyro-
have been in different
KHAN
TABLE
1.
Titers
of Leptospira
interrogans
Number (%) at
Serovar
Autumnalis
positive 1:50
antibodies
in sera
of
wolves
50
in
of sera
Number
.
ET AL-LEPTOSPIROSIS
100
IN WOLVES
249
Minnesota.
sbowing
titerb
200
of
400
800
>1,600
15 (3.3)
5
1
4
1
2
2
3 (0.7)
2
1
0
0
0
0
Bratislava
18 (3.9)
14
1
2
1
0
0
Canicola
13(2.8)
5
3
3
0
0
3 (0.7)
3
0
0
2 0
0
0
24 (5.3)
9
4
6
3
1
1’
Hardjo
2(0.4)
1
1
0
0
0
0
Pomona
7(1.5)
2
1
3
1
0
0
Pyrogenes
7 (1.5)
1
1
3
1
1
0
9
4
3
Ballum
Copenhageni Grippotyphosa
92 No
serum
was
positive
for
Reciprocal
of highest
One
serum
was
positive
This
serum
had
a titer
‘Of
sera
457
of positive
species
examined, sera
of
tional States 50010,
australi.s,
dilution 1:1600
of
1:1600.
52
were
appears
hosts
(Fame, 1982). were obtained
serovars
serum at
42
(20)’
the
positive
and
batavlae,
showing and
13
50% other
at
at
1:6400.
1:50.
Because
the
world
some
in
various
parts
in semi-solid
of
had
antibodies
to more
than
one
serovar,
the
number
RESULTS
these bacteria media from the Na-
cultures
of
Veterinary Services Laboratory (United Department of Agriculture, Ames, Iowa USA). These organisms were sub-cul-
test.
The microscopic agglutination at., 1979) was performed in 96-well,
test
microtiter McLean,
Laboratories,
(Linbro; 22102,
Flow USA).
(Cole
et
flat-bottom Briefly,
a
Of
457
sena
examined,
52
positive at titers of were positive at titers
laboratory. The stock cultures were maintained in semi-solid media and liquid cultures were used as sources of antigen for the microagglu-
1:25
dilution of test serum was made in phosphate buffered saline, pH 7.2. To 50 j.il of this diluted serum was added 50 tl of live antigen at 25 nephetometenic turbidity units (NTU) making a final serum dilution of 1:50. The plates were incubated at 29 C for 120 ± 30 mm and examined for agglutination under a dark-field microscope. The samples found positive on screening at a 1:50 dilution were further titrated using serial two-fold dilutions of up to 1:6,400. We considered microagglutmnation titers of 1:50 as positive. Data were analyzed via a Chi square test with Statistix software (N.H. Analytical Software, St. Paul, Minnesota 55104, USA, 1985) and an IBM personal computer (IBM, PC, International Business Machines, Rochester, Minnesota 55901,
USA).
sera
to be 92.
Stock
plates Virginia
tarassovi.
agglutination.
tured in both semi-solid and liquid (bovine atbumin-polysorbate 80, Intergen Company, Purchase, New York 10577, USA) media in our
tination
21
bodies against interrogans. itive
for
ben
of
one Because
more
in Table
to more 2 serum
,
more some
one
senovar sera were
52 positive
of L. pos-
the
is shown
than one samples
were
31 (6.8%) for anti-
senovan,
samples
1. Of the
positive 1 1 and
1:50, and of 1:100 or
than
positive
(1 1 .4%)
num-
to be
92
sena,
25 were
serovar. contained
Twelve, anti-
bodies against two, three, and four serovans, respectively. At titers of 1:50, the prevalence of antibodies to serovans in decreasing
order
bratislava nicola
was:
(3.9%), (2.8%),
(1.5%),
ballum
and was
hardjo positive
viae,
and
grippotyphosa (1.5%),
(0.7%),
copenhageni
tarassovi. to
(3.3%),
pomona (0.4%). None for senovans
antibodies
The
different
1:50-400
for bratislava, and
hard
Jo. Antibody
penhageni Of
wolves
range
in titers was:
1:50-1,600 for pyrogenes;
sex
at was
and
ballum
against
positive
whose
samples bata-
senovars
for
titers
were
(0.7%),
the australis,
canicola
1:50-100
Ca-
pyrogenes
of
50-6,400 for autumnalis; grippotyphosa; 1:50-800 mona;
(5.3%),
autumnalis
serovar 1:50
of 1: for Poand co-
only.
known,
212
250
JOURNAL
TABLE
2.
OF WiLDLIFE
Distribution
DISEASES,
VOL. 27, NO. 2, APRIL
of Leptospira
1991
antibodies
interrogans
in wolves
Number Number County
Number
tested
Lake
positive
181
Bal-
Brati-
nalis
lum
slava
3
0
4
1 1 1 1
0 0 2 0
0 2 4 1
15(8.3)
Minnesota
a t iter
sh owing
Autum(%)
from
of
according
>1 :50
to counties.
against
Canic-
Pomo-
ola
Pyro-
Grippo
Hardjo
na
genes
8
2
0
0
1
0 1 0 1
1 4 7 1
0 0 0 1
0 1 1 1
0 1 0 0
Koochiching St. Louis Kittson
9 22 20 62
1(11.1) 5 (22.7) 7 (35.0) 3(4.8)
Itasca
11
3(27.3)
1
0
1
0
2
0
1
0
2
0(0.0)
0
0
0
0
0
0
0
0
8
2
11
10
17
1
4
2
Beltrami
Roseau Total Some
307b
of the animals
Excludes
were
samples
mates
showed
titers
for which
county
and
of seropositivity for females statistically in the rates
34 (11.2)
198 for
to two
were
of origin
was
females.
mates
was
serovars. unknown.
The 11.6%
rate
found
and
ka
it was 12.3%. There was no significant difference (F> 0.05) of seropositivity between males
and
females. Wolves were
ties
in northern
collected
from
Minnesota.
seven
of seropositive wolves near (20.1%) was 2.6 times greater wolves living in wilderness (7.7%)
(Table
coun-
Prevalence
antibody positive wolves from ties is shown in Table 2. The
farms
or more
of
these counprevalence
farming than away
areas that of from
3).
DISCUSSION
The 11.4% seroprevatence study is lower than
in wolves observed
in by
only
1 of 80 wolf
positive
Presence
for of
L.
samples
antibodies
in
dicate previous or current may have resulted either tact with urine of other eating have
infected indicated
may
Alas-
antibodies. wolves
may
in-
infection which from direct conwolves or from
prey. Reilly et that oral infection
idae and other carnivores rogans is possible. They interrogans
from
interrogans
a!.
with observed
be protected
L.
(1970) of Caninterthat L.
from
gastric
acidity because the gulped bolus passes partially digested into the alkaline duodenum. Once infected, wolves may act as maintenance hosts for L. interrogans on account of their highly social behavior.
Everard animals examined
et a!. (1983) in 31 species of wild from Trinidad and Grenada. They 894 animals and found 198
Leptospirosis is endemic in bovine, percine and equine populations of Minnesota. Of 17,014 animal sera examined from 1984 to 1986 at the Minnesota Veterinary Diagnostic Laboratory (St. Paul, Minnesota
(22.1%) results,
positive however,
Our to
55108, against
who
rogans at titers of of antibodies against
our
those
of
Zarnke
that
at titers are in and
of 1:100. sharp contrast
Ballard
(1987)
TABLE 3. Relative prevalence of antibodies against Leptospira interrogans in wolves depending upon their proximity to dairy and beef cattle farms.
Wolf population
Seropositive
at 1:50
Preva-
Sero-
negative at 1:50
lence Total
(%)
27
107
134
20.1
related
25
298
323
7.7
Total
52
405
457
Farm
related
Non-farm
USA), 3,040 (17.9%) had antibodies one or more serovars of L. Inter1 :50. The different
prevalence serovars
in
decreasing order was: icterohaemorrhaglae (24.9%), hardjo (2 1 .4%), canicola (20. 1 %), pomona (18.5%), and grippotyphosa (15.1%). It is possible for a particular serovar to be shared by many maintenance hosts or to be specific to a single host (Hathaway highest for study
et a!., 1983b). seroprevalence
serovar
In our study, (5.3%) in wolves
grippotyphosa.
of moose
in Minnesota,
In
the was
a previous Diesch
et al.
KHAN ET AL.-LEPTOSPIROSIS
(1972) found that var grippotyphosa 328 (27.1%), white ovar pomona were
the prevalence of serowas the highest, 89 of antibodies against serthe least prevalent (18
of 328) (5.5%). Serovar grippotyphosa many countries including China mitsu
(Tonkonozhenko et at., 1987)
from dog, zibethicus),
cattle, swine, squirrels
striped
red fox ( Vu! pes cinereoargenteus), us)
and
opossum
(Hanson,
1982;
species of interrogans
(Ondatra niger,
(Mephitis
vu! pes) hare
,
S.
mephitis),
(Diadeiphus et at.,
may organisms
be
36 (31%)
were (47%)
53
gray
had titers
foxes
Two animals hard Jo albeit 1:100. Collins
most foxes
having
prevalent and 11 of
titers.
Juvenile
higher
geometric
were positive to serovar at tow titers e.g., 1:50 and et at. (1981) have reported
the presence of antibodies Jo in pronghorns (Antilocapra
against
may
indicate
sometime subsequently in L. titers
existence
earlier
the
and decreased.
interrogans may mean
cination residual mona infection
that
persist or longer of
that
were their This
infected
titers have is common
infections where high active infection or vac-
and a fall infection. can
wolves
in titer may indicate Titers to serovar Poin deer (Ferris
antibodies
for 3 mo et a!. 1960). to
multiple
after The sero-
serovars. in wolves was 20.1%
to 7.7% prevalence in wilderis not surprising because L. in(especially serovar pomona)
has
isolated
been
such farm
from
as in recreational waters (Diesch
where
it has
long time. vived for taminated
been
found
of infection
to
survive
and 1966) for
to wild
animals.
It was
disinfection of break the chain to wild animals,
livestock of infecdomestic
and
rodent populations (Will and Contrary to this opinion, it postulated that wild animals may
demiology need for mine the
of L. interrogans infection cattle. However, conclusive area of L. interrogans epi-
is not available indicating more detailed studies to actual rote of wolves and in spreading
and
tospirosis in domestic and elsewhere.
the deterother
maintaining
animals
lep-
in Minnesota
ACKNOWLEDGMENTS We tance,
thank Mary W. K. GomI,
Thurn for secretarial M. E. Nelson, W.
assis-
J. Paul, and many volunteer technicians for assisting with blood-sampling, and U.S. Seal for serum storage. This study was funded by the U.S. Fish and Wildlife Service and the U.S.D.A. North Central Forest Experiment Station. LITERATURE T. C.,
AMUNDSON,
La
AND
virus
Crosse
CITED
T. M.
infection
YUILL. in the
1981. red
fox
Natural (Vulpes
fulva), raccoon
gray fox (Urocyon cinereoargenteus), (Procyon lot or), and opossum (Dideiphis virginians). American Journal of Tropical Medicine
ARMITSU,
a
surconwhich
1972).
act as sources for deer and proof in this
wildlife
environment
(1971) to believe that manure farm area was a potential pubproblem and could also be a
suggested that wastes may help tion from farms animals, Diesch, has been
the
waters, ponds, and McCultoch,
For example, teptospires 61 days in experimentally oxidation ditch manure
L. hardamericana)
of Colorado. Antibodies against serovar pomona, which is a common serovar in domestic animals, were also tow in our study. The tow antibody titers in wolves
with
compared ness. This terrogans
source
Many with L. 1981).
in red and gray of southwestagainst serovar
significantly than adults.
various
251
especially cross-reactivity
The prevalence of antibodies mnhabitating farming areas
virginianus) infected (Shotts,
the red
among
1971).
and Yuill (1981) examined the of antibodies to five different
grippotyphosa with 25 of
of L. interrogans, titers, may indicate
led Diesch in the open lic health
gray fox (Urocyon (Lepus american-
serovars of L. interrogans foxes (U. cinereoargenteus) ern Wisconsin. Antibodies
foxes mean
in and
(Lynx rufus), cottonfloridanus), swamp raccoon (Procyon lo-
Shotts
foxes
Amundson prevalence
muskrat (Sciurus
,
skunk
prevalent U.S.S.R.
et at., 1965; Arhas been isolated
and
carolinensis), bobcat tail rabbit (Sylvilagus rabbit (S. aquaricus) tor),
is the
types tow
IN WOLVES
and Y.,
Hygiene T.
30:
MATUHASI,
706-714.
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T. SATO,
252
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