Mann-Whitney non-parametric rank analysis ...... KEYES. 1977. Naturally occurring leptospirosis in northern fur seals. (Callor/zinus ui-sinus). Jour- nal of. Wildlife.
J(fl4fl1(ll ‘f 5%ik//if.
:32(4
J)iaas,s
© \VjkIIife
LEPTOSPIROSIS
IN CALIFORNIA
CALIFORNIANUS) COAST,
STRANDED
The Marine Department
and
Society
School
of Veterinary
Wildlife
Pathology
nianu.s)
Medicine,
stranded 1994.
Clinical these
stains
of
sections
pomona of 20
than
animals
sampled
Leptospirosis
is
1984,
1988, In
20
titers
to
a cosmopolitan
has
caused
zootic and California
enzootic sea lions
disease
and
both
served (USA)
along coast,
the and
Leptospira
to
isolated
from
the
kidney
and
of ob-
and
(Dierauf
et
a!.,
on
the
coast
1985).
failure
urine
of
Channel and
premature
pups
idence
that
in
sea
The enzootic supported
nature by the
a
1972
and
may
consistently
lions
(Smith
sea the
of L.
leptospirosis
in California
was
off
isolation
titers
of
in
1993
due
to
the
antibodies
to document
three
in sea licoast, and over
December
lions
to
gross
of disease
to
to
and
characteristic
pattern
14 yr.
1994,
admitted
to
as
ob-
a rehabili-
center. MATERIALS
Stranded the and
31
their
from
1973
was
be
enzootic
et al.,
sea lions between 1984 and et al., 1993). In animals with
disease,
L.
sexes
ternal
mated sagittal
ev-
tion
1974).
der
utilizing crest
standard development
body and
1978).
(Bossart
placed
renal
Animals
and samples
vein
animals
older
were
using and
into
juveniles,
an
taken 18
Dierauf,
vacutainers
the
rimni 572
separation
than from X
1990)
and
containing and
clot
of
activator
exesti-
weight, colora-
age
4 to
acid
gel
ages
length, pelage
gauge
ylene-diamine-tetra-acetic
of
their
3 yr
as
clinically,
examination and
classed
a reha-
examined
by
this
Mammal
California,
were
to 1981
in
Marine
morphology
(Mate,
Blood
Sausalito, they
Januarv
included
The
determined
were
teal
were
at
along
123#{176}05’\V 1
genital
subadults,
1990
1994
facility,
appearing
between
arrival
(TMMC),
bilitation
lions
(37#{176}42’N,
121#{176}:30’\V)
Upon
METHODS
sea
coast
December
study.
re-
AND
California
California
35#{176}59’N,
California
considered
the
af-
of the disease was also fact that renal disease common finding in
was
titers
disease,
were
in sea
tation
lions
pomona
stranded (Gerber
pomona
of
high
1981
Center
Islands
the
of
California
antibody
was
Observations
in
were obEpizootics in the autumn,
had
spp. and findings.
review
served
fected animals (Vedros et a!., 1971). A second epizootic was observed in central California and Oregon in 1984, and clinical progression of the disease was described productive
organisms in 1991.
common
cause
545
impregnation
seronegative,
objectives
January
Oregon
pomona
exaniined;
silver
further epizootics of !eptospirosis ons along the central California
californi-
California
In
more were
morteni
and
animals
sampled
of
Our
epi-
large numbers sea lions were
were
animals
likely
post
free-living
in
(Zalophus
1970, unusually dead California
and 47
calforDecem-
(Zalophus 1981
spiral sanip!es
kidne
presence Lepto.s’pira
of both do(Shotts, 1981).
variety
of 2338
coiled
four
1994,
lions
January
of nephritis.
animals sampled L. pomona.
disease
sea
(33%)
loosely
most
pomona
In
764
1994,
USA
USA
between
lesions from
and and
1992,
low
in
numerous
1991,
T. Spraker4
94965, USA. California 95616,
USA
Calif’ornia
coast
gross
and
California at Davis,
80525, in
cultured
1991
In
had
Leptospira
L. Morgan,1
95616,
Colorado
seen
similar was
1:3200.
of humans and a wide mestic and wild animals
sick
CALIFORNIA
USA
(USA)
kidney,
males.
greater
determined
INTRODUCTION
anus).
CENTRAL
California
was were
with
kenniwicki in
Davis, Collins,
California
fornialin-fixed
juvenile
92112,
Fort
disease
died,
occurred affecting
central
renal
pomona
leptospirosis
three
THE
A. Colagross,3
California
of California,
Stanford,
the of
of
Diego,
leptospirosis
along animals
Leptospira
typically
2905
signs 764
San
University
of
live
of
L.
1996
(ZALOPHUS
Gate National Recreation Area, Sausalito, and Immunology, University of California
Diego,
International,
(71%)
of
of San
Prevalence
ABSTRACT:
served.
ALONG
L. J. Lowenstine,2
M. Koski,1
Mammal Center, Golden of Pathology, MicrObiology
Zooloogical
ber
LIONS
pp. 572-550
Association
1981 -1994
F. M. D. Gulland,1 2
SEA
1996.
Diseas,-
and
sun-
5 yr-olds
as
5 the
yr
38
mm
as
adults.
caudal
gluneedle
aliquots
were
either (EDTA)
ethor
(Vacutain-
Se-
GULLAND
Becton Dickinson, Rutherford, New Jersey, USA). Complete blood counts were performed on EDTA samples using a Coulter Counter Mode! S-Plus IV (Coulter Electronic Inc., Hialeah, Florida, USA), differential cell counts were performed manually (Brown, 1980). er,
Tubes
containing
serum
were
centrifuged
at
3,000 X G within 2 hr of collection, the serum separated, and aliquots frozen at -20 C for fluture serological analysis. Serum biochemistry analyses were performed on a Vet test 8008 (Idexx Laboratories mc, Westbrook, Maine, USA) and serum electrolytes were measured on a VetLyte Electrolyte Analyser (Idexx Laboratories mc). Microagglutination tests using Leptospira pomona antigen (Galton et al., 1962) were performed at the California Veterinary Diagnostic Laboratory Service, Davis, California, on stored serum. Samples for serology were taken from 27 aninia!s that died of renal disease in 1991 and 20 animals in 1994. Five of the latter were sampled twice, 14 days apart. Microagglutination tests were also performed on serum samples from 53 clinically normal juvenile animals that stranded in 1992 and 1993. Post mortem examinations were performed on
a!!
Following
samples colon,
animals
that routine
of lung, pancreas,
gonad, urinary were fixed by fered forma!in. in TissuePrep
died post
heart, spleen,
bladder, immersion Fixed (Fischer
during mortem
thyroid,
rehabilitation. examination,
stomach,
liver, kidney,
ileum, adrenal,
lymph node and brain in 10% neutral buftissues were embedded Scientific, Fairlawn, New Jersey, sectioned at 5 rim, and stained with hematoxylin and eosin. In 1991, slides of fixed kidney tissue from the 27 animals that were tested serologically for !eptospirosis were stained with Warthin Starry silver stain (Luna, 1968). In 1994, slides of kidney from 10 animals that died with gross lesions of renal disease were stained with Steinart stain and Levaditis stain (Luna, 1968). Samples of liver and lung from all animals examined at post mortem between January 1990 and December 1994 were cultured on blood agar with 5% added citrated sheep blood, and on MacConkey agar (PML, Tualatin, Oregon (USA)) incubated at 35 C, then examined at 24 and 48 hr (Carter, 1973). Bacteria were identified using the API 20E System (Sherwood Medical, Plainview, New York, USA) and colony and biochemical characteristics (MacFaddin, 1980). In 1991 and 1994, Leptospira spp. isolation was attempted from kidney and urine from 87 and 20 animals respectively. In 1991, 2 to 3 g samples of kidney from animals with gross lesions typical of leptospirosis were aseptically ground in 1% bovine serum albumin
ET AL.-LEPTOSPIROSIS
IN SEA
LIONS
573
(BSA) and the tissue debris allowed to settle. The supernatant was filtered through a 0.22 pm mil!ipore filter and was used to inoculate Fletchers and Ellinghausen. McCullough, Johnson and Harris (EMJH) media (Collins et al., 1995). Urine samples collected from 12 of the same 87 animals were centrifuged at 600 X G for 10 mm., the supernatant removed and the pel!eted material added to 1% BSA. Three serial tenfold dilutions (1:100, 1:1,000, 1: 10,000) of the suspended material in 1% BSA were prepared and 1 ml of each dilution used to inoculate EMJH media. Inoculated tubes were incubated at 28 to 30 C and examined at weekly intervals using darkfield microscopy. The resulting four isolates were serotyped at the National Veterinary Diagnostic Laboratory (NVDL), Ames, Iowa (USA) (Fame, 1993). In 1994, twenty 2 to 3 g kidney samples were shipped
to
the
Laboratory,
Fletcher’s In
Colorado
Fort
media
1991,
Veterinary
Collins,
and
cultured
impression
of
smears
0.05.
A
sea
(USA),
in
as described. were
made
from
gross lesions typical of leptospirosis 24 hr of death. Smears were also made from 52 of the 104 kidney specimens after they had been frozen at -20 C for greater than 4 wk. Smears were airdried, fixed in acetone for 10 mm, then examined for Leptospira spp. using a direct fluorescent antibody technique (Galton et a!., 1962). Hematology and serum biochemistry parameters were compared to normal ranges (Roletto, 1993) using a one sample t-test (Ott, 1993). Sex and age differences in prevalence of leptospirosis were compared using the chi-square test with Yate’s correction factor (Ott, 1993). Statistical significance was determined at P < kidneys
104
Diagnostic
Colorado
lions with within
Mann-Whitney
analysis
non-parametric
(Statview
4.01,
Abacus
Berkeley, California) was numbers of sea lions that and the percent prevalence, to non-epizootic years.
used died
rank Concepts,
to compare the of leptospirosis, in epizootic years
RESULTS Between ber ed
1994, live
1 January 2,338 along
the
and
were
admitted
764
(33%)
had
ing
depression,
of
similar
a
pain hunched
clinical
was
strand-
signs
use
the
these, includde-
hindflip-
vomiting,
abdominal determined
position
coast Of
po!ydipsia,
cases,
and
Decem-
TMMC.
to
in extreme
31 lions
California
to
tremors,
dominal
central
reluctance and,
cular
and sea
anorexia,
hydration, pers,
1981
California
by and
muspain;
ab-
adoption
holding
the
574
JOURNAL
TABLE
1.
OF WILDLIFE
Mean
leptospirosis
(±
confirmed
with
animals
clinical
SI)) b signs
DISEASES,
VOL.
32, NO. 4, OCTOBER
senum biochemical values observation of spirochete-like resembling leptospirosis.
157
(nsEqll)
Calcium Blood
Nitrogen
Creatinine Roletto
I) These
values
Marine
are
front
15 clinically
158
8
149
±
2
4.2
±
2
4.6
±
0.4
9.3
±
1.6
9.2
±
0.8
9.4
±
0.5
14.2 ± 5.4
1:3.8
±
2:3
5.9
±
1.06k’
over
blood
the
±
57
182
±
:31
44
±
20
7.6
±
4.6
8.1
±
1.5
0.7
±
0.2
diuin
and
Five
Statistically
urea
764
gross
lesions
These
were
significant
levels
animals
died,
the
torntal
ramtge
severe
2).
thickening
branes
127
from
to
neys,
marked
loss
of
medullae
of
was the
glomerular
characterized
basement
cortices
and
orrhages
of pale
occasionally
and
necrosis
of
the
..,
ren-
tan
hemorrhage
renal the
junction
at
lesions, animals
(Fig.
tenacious
severe
gastric
bile
friable
in the
ulceration.
gall
1.
...
‘5 .3
1
.
. .
to
.,i-.
of
livers
with
bladder
On
%.
cortico-
addition one-third
swollen,
.
#{182}5
hem-
the In
approximately had
thick,
1).
..
I
colored
subcapsu!ar
..
kid-
.#{149}.5 .5.
medu!lary
mem-
capillaries,
similar
between
cortices,
multiinterstitial
This of
animals,
examination.
swelling
differentiation and!
at The
chronic,
lymphoplasmocytic (Fig.
mild
tissues
subacute
:;.,
ule
exaittimed
1).
had
mortem
focal, by
so(71%)
and!
of
observed
nephritis dc-
(Table
forty-five
post
of
phosphorus,
and on
within
examination
we
367
included
nitrogen,
creatinine
signif-
detected
from
changes
hundred!
these
l)ltXXl parattiters
ss-ithu other
No were
collected
animals.
1)100(1
animals
abdomen.
biochemical
vated
healthy
changes
samples 764
serum
176
in 1994.
hematologic
these
SO)
1.3
(mg/dl)
Center
hindflippers in
salne
=
(199:3).
Mamnnual
icant
nec (it
16
(mg/dl)
From
Hufere
±
(mg/dl) Urea
I .eptospirt)sis stuspect (ii = 357)
with and in
±
(mg/dl)
Phosphorous
sea lions (/a-zlopIzus (‘a!fi)nuiai1uz) silver-stained sections of kidne
in
4.14
(mEq/l)
Potassium
California organisms
1.eptospirosis ct)mmfirmmteci (it = 20)
Parameter
Sodium
in
1996
5,-I..... ‘S..
and
histological
I’
‘i1’’ - S..
‘
,r..,
. ‘2
‘1.
FIGURE
1.
leptospirosis. Bar
=
2 cm.
Kidney Note
from severe
a California loss
of
renule
sea
lion with
definition.
FIGURE pisoplaxmnocytic and eosin
2.
stain.
Cdifornim interstitial Bar = 10()
sea
lion nephuritis. l.u
kidney
with I lemnatoxvlin
lviii-
GULLAND
ET AL.-LEPTOSPIROSIS
2.
TABLE
Antibody
IN SEA
prevalence
spira pomona in California fornianus) between 1991
and
PosiYear
..‘1
Number
live
sampled
(%)
Antibod 1:3,200
100
575
titers to Lepto(Z.alophus cali-
and lions 1994.
sea
LIONS
y titer
1:800
1:4(X)
1:1(X) 0
1991
27
0
0
1992
33
15
27 3
0
1
1
1993
20
15
0
1
0
2
1994
25
0
0
0
100
25
..
.1i.,
#{149}‘ i.’:
:
S
erichia
Eighty-nine tibody tests
d
all
negative.
Slides
ground
L convoluted
mulation
in
Levaditi’s
a California
sea
and
plasma
debris,
52
and
stains
on were
In
kidney
ganisms
sections
thin,
the
cultured
kidney
in
sembling the
1991,
spirochete
Leptospira
spp.
kidney on
and
darkfield
Organisms
and
terrogans,
three isolated
by
lated
from
20
1994.
Additional
kidney
samples
No
in
all
urine
samples
the
seen urine
from
four strain
spirochetes samples
were
1:3,200,
33
spp.
Cases
cultured
from
all
included
Esch-
but
of
had
both
occa-
had
titers 12
in
had
greater
had
in
similar
titers
from
1992
and
had
antibodies
1993,
titers
five
three to
of Lep-
were
typical at
all
between
years 1982.
lower
1988,
1991
(Dierauf cases
(Fig.
numbers
of
sea
California.
Peak
September
in in
1984,
1994.
al., the
(P
(Fig.
lions
1988
In
each
July
season
and
when
strand
in
were were
The docu-
1985).
and
< in
3).
between
numbers Cases
and
observed
previously
seen 4),
ex-
1981
1994
been et
were
!epto-
mortem
were
and has
of
post
Significantly
prevalences
epizootic
October
disease
observed than
1984 year,
renal in
1984,
est
on
Samples
1993
were
December in
18
animals in
mented
iso-
and
an-
intervals
icterohemorrhagiae
bratislava.
of
other
bacteria years
than
higher
cultured
five
greater
1994
kenni-
The
1994
0.01)
in-
2).
14 day
pomona
from
12
kidney
Leptospira
(Table
and
L.
1:3,200
resam-
to
than
amination
on
titers
with
1991
2).
spirosis
examination. as
kidney
in
in
animals
animals
(Table
disease
Fifteen
L.
tospira or-
organisms
pomona,
NVDL.
(15%)
3).
were of
identified
serovar
wicki
12
microscopic
were
samples,
and
in-
cells.
greater
L.
were back-
animals
at
gryppo,
were
ruptured from
1:3,200
1:3,200,
against
animals,
and renal
anwere
excessive
twice
to
than
spiral
(Fig.
L.
20
spirochetes
examined
87
37
coiled
with
samples
Of
from
sions.
of
impregnation
loosely
compatible
kidney
collecting within
silver
than
titers
samples
to
antibody
sampled
kidneys
frozen
samples of
had
titers
accumulation
lymphocytes
tissues.
there
bacteria
and
moderate
and
fluorescent in 1991
frozen
due
serum
imals
to
read
signs
greater
lion.
accu-
and
tubules
a
cells
terstial
epithelium,
cellular
convoluted
ducts
ples
material
from
tubular of
within
of
stained
of kidney
100
=
of
Silver
on from
fluorescence
1994 3.
tests
to
clinical
section
Kiebsiella
mirabilis,
of the 104 fresh kidneys
on
positive,
All
FIGURE
sp.,
Proteus
‘3
S.
.f
#{149}
/
Bar
sp., sp.
difficult
stained
Enterobacter
Salmonella
Pseudomonas
:..
.
coli,
sp.,
highcentral seen
1991, most
in
and corn-
in
576
JOURNAL
OF WILDLIFE
DISEASES.
VOL.
32, NO. 4, OCTOBER
1996
I
#{149} Juiy
#{149} August
40 ii C 0 30
U
September
0
October
0
November
9
December
V V 0
E
z 1984
1991
1988
1994
Year Year FIGURE
4.
amined
Number
post
with
of
mortem
typical
at
lesions
December
California
The
of
5.
FIGuRE sea
Marine
lions
Mammal
leptospirosis,
ex-
1981
for
both
sex
male
and
animals
age
class)
(P (Fig.
