J.
Helminthol. Soc. Wash.
64(1),
1997
pp.
137-141
Microfilariae
in the
Free-Ranging
Florida
Panther
(Felis
concolor
coryi)
MARNIE
G.
LAMM,'
MELODY
E.
RoELKE,
2
-
5
ELLIS
C.
GREiNER,
3
-
6
AND
CHRISTINE
K.
STEiBLE
4
1
College
of
Veterinary Medicine, University
of
Florida, Gainesville, Florida
32610
(e-mail:
v968908@student.health.ufl.edu),
2
Florida Game
and
Fresh Water Fish Commission, Gainesville, Florida
32610,
3
Department
of
Infectious Diseases, College
of
Veterinary Medicine, University
of
Florida, Gainesville, Florida
32610
(e-mail:
greiner.vetmed3@mail.health.ufl.edu.),
and
4
Department
of
Statistics, Institute
of
Food
and
Agricultural
Sciences,
University
of
Florida,
Gainesville,
Florida
32611
ABSTRACT:
Blood samples from Florida panthers (Felis concolor coryi) collected
from
1986
to
1993 during
the
months
of
December through
May
were screened
for the
presence
of
microfilariae
(mff)
by the
Difil®
filter
test.
Thirty-five
of 47
(74.5%) panthers older than
2 yr of age
were positive with
microfilaremias
ranging from
10
to
7,380
mff/ml
of
whole blood.
No
panthers that were
6 mo of age or
less
(n
= 10)
were
microfilariae-
positive,
and
only
20% of the
panthers
in the
1-yr
class
(n = 5)
were positive.
A
representative number
of
microfilariae
(n = 40)
from
each
of 7
freshly
collected positive blood samples
was
measured
and
morphological
characteristics were noted.
The
average length
of
microfilariae processed
by the
modified Knott's technique
was
320
/Am
(273-370
/Am)
with
a
width
of 4-5
/Am.
Of the 280
microfilariae measured,
202
(72.14%)
had
tapered
heads
and
straight tails
with
an
average length
of 319
/im
(276-368
/Am),
61
(21.79%)
had
blunt heads
and
straight
tails
and
averaged
323
/urn
(274-366
/Am),
16
(5.71%)
had
tapered heads
and
button-hooked tails with
an
average
length
of 320
/Am
(290-368
/am),
and 1
(0.35%)
had a
blunt head
and
button-hooked tail
and
measured
320
/Am.
The finding of no
significant
difference
(P >
0.05) between length measurements
due to
differences
in
head
and
tail shape leads
us to
believe that
all
microfilariae were
of 1
species. Based
on
micro-
filarial
length
measurements, review
of
necropsy reports,
and
comparison with
bobcat
microfilariae,
the
most
likely
filarial
species infecting
the
Florida panther
is
Dirofilaria
striata
(Molin,
1858).
KEY
WORDS:
Florida panther, Felis concolor coryi, Dirofilaria
striata,
microfilariae, morphology, prevalence,
bobcat, Lynx
rufus.
Filarial nematodes have been reported
in a
number
of
exotic
felid
species. Dirofilaria
re-
pens
has
been reported
in the
lion (Panthera
leo)
(Nelson
et
al.,
1962). Dirofilaria
immitis
has
been found
in
jaguars (Felis onca), tigers (Felis
sondiacus
and
Felis
tigris),
wild cats (Felis
bangsi
costariensis),
jagouarundi (Felis
yago-
uarundi)
(Otto, 1974),
a
bengal tiger (Panthera
tigris) (Kennedy
and
Patton,
1981),
and
bobcats
(Lynx
rufus)
from
Florida (Levine, 1980; For-
rester, 1992).
Of 22
free-ranging mountain lions
(Felis
concolor) from California,
1 was
seropos-
itive
for
antibodies against
the
somatic antigen
of
D.
immitis
yet was
seronegative
for
cuticular
antigen
and no
circulating microfilariae (mff)
were found
in the
whole blood (Paul-Murphy
et
al.,
1994). Dirofilaria striata
was first
reported
in
Brazilian pumas (Felis concolor
and F.
ma-
croura)
(Raillet
and
Henry,
1911)
and
since
has
5
Present address: Tanzania National Parks,
Arusha,
Tanzania, Africa.
6
Corresponding author.
been found
in the
ocelot
(Felis
pardalis),
the
margay (Felis
tigrina)
(Anderson
and
Diaz-Un-
gria, 1959),
and the
bobcat (Orihel
and
Ash,
1964; Miller
and
Harkema,
1968; Roelke,
un-
publ.
1985). Forrester
et al.
(1985) reported
adult
D.
striata
in
mff+
adult Florida panthers.
The
Florida panther
is an
endangered subspe-
cies
of
cougar that inhabits pockets
of
habitat
in
the Big
Cypress
and
Everglades ecosystems
of
southern
Florida (U.S.A.).
A
population
of
less
than
40
adult animals remains
in the
wild (Bel-
den, 1986). Since veterinary involvement
in the
Florida Panther Recovery Program began
in
1983,
an
intensive health evaluation
and
moni-
toring program
has
been
in
effect.
The
protocol
includes
the
collection, screening,
and
storage
of
various biological samples each time
an
animal
is
immobilized.
In the
past,
the
presence
of mff
has
been noted either
by
buffy
coat analysis,
by
membrane
filtration, or in
reports from clinical
laboratories. Adult
D.
striata were found
in 4
out
of the 7
panthers examined
from
1973
to
1983,
3 of
which were
mff+
(Forrester
et
al.,
137
Copyright © 2011, The Helminthological Society of WashingtonCopyright © 2011, The Helminthological Society of Washington
138
JOURNAL
OF THE
HELMINTHOLOGICAL SOCIETY
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JAN
1997
1985).
It was
assumed
that
all
microfilariae
from
these
animals
were
representative
of D.
striata
because
no
other
adult
filariids
were
detected.
Adult
D.
striata
were
found
in 5
additional
microfilaremic
panthers
at
necropsy
(Roelke,
Nayer,
and
Vickery,
unpub.
data
1985).
Accord-
ing
to
necropsy
reports,
1-3
adult
filariids
were
present
singularly
in the
fascia
between
muscle
bundles
in the
distal
extremities.
No
reports
of
any
filarial
species
other
than
D.
striata
were
found
in
review
of
Florida
panther
necropsy
re-
ports
(n = 41)
from
1980
to
September
1994.
This
study
was
undertaken
to
determine
the
prevalence
and
microfilaremia
of
Dirofilaria
sp.
in
the
Florida
panther
population
and to
char-
acterize
and
measure
the
mff
in
order
to
confirm
the
identity
of the
species
of filariid
present.
Materials
and
Methods
From 1986
to
1993,
a
total
of 83
blood samples
representing
47
individual
panthers
and 3
bobcats
was
screened
for the
presence
of
mff. Samples were col-
lected during
the field
capture season
in the
months
of
December through
May
from free-ranging panthers
in
the
Big
Cypress
and
Everglades ecosystems.
All
ani-
mals were anesthetized with ketamine hydrochloride
(Ketastat®,
Bristol Laboratories, Syracuse,
New
York,
U.S.A.)
or a
combination
of
tiletamine/zolazepam
(Tel-
azol®,
A. H.
Robins Co., Richmond, Virginia, U.S.A.).
Blood
was
drawn prior
to
administration
of fluids,
vac-
cinations,
and
prophylactic injections
of
Ivermectin
(Ivomec®,
Merck
&
Company, Incorporated, Rahway,
New
Jersey, U.S.A.)
at a
dose
of 200
meg/kg.
Blood
samples were taken directly from
the
saphenous vein
into
sterile
3-ml
tubes containing ethylenediaminetet-
raacetic acid (EDTA)
via a
vacutainer
and
butterfly
apparatus.
The
sample
was
kept
cool
in an
insulated
container
for the
2-8-hr
transition from
field to
lab.
One
milliliter
of
EDTA preserved whole blood
from
each
of the 17
samples collected
in
1992
and 5
sam-
ples from 1993
was
processed using
the
Difil®
(EV-
SCO
Pharmaceuticals, Buena,
New
Jersey, U.S.A.)
procedure described
by
Howland
and
Todd
(1977).
In
addition,
58
whole blood samples preserved
in
EDTA
that
had
been collected from free-ranging panthers
from
1986
to
1991
and
stored
in a
1:10
ratio
of
blood
(1
ml) to 2%
formalin
(9 ml) at
4°C
were screened
for
mff.
From
the
10-ml
sample,
5 ml
were passed through
a
Difil
filter.
Numbers
of mff
were determined
for all
positive
samples (except
FP#5
1 in
which high numbers
of
mff/field
made accurate counting impossible)
by
counting
all
individual
mff on the filter at a
viewing
magnification
of
X200
to
obtain
a
mff/1
ml
value.
When no mff were found, the remaining 5 ml were
tested
in the
same manner
to
confirm
that
the
entire
sample
was
indeed negative.
In all
cases,
the
Difil
filter
holder
was
washed
and
dried well between samples
in
order
to
avoid cross-contamination leading
to
false-
positive results.
Of the
17
samples screened
in
1992,
7
samples
with
high
numbers
of mff
were chosen
for
morphological
study
of
individual microfilariae.
All
blood
samples
were kept
in
EDTA-coated blood tubes
for the 2-8 hr
between time
of
collection
and
time
of
microfilarial
analysis.
A
1-ml
aliquot
was
processed
using
a
modi-
fied
Knott's procedure (Knott, 1939).
Microfilariae
were measured immediately after processing
in
order
to
minimize possible
effects
on
length
or
width
of mi-
crofilariae
due to
storage
in 2%
formalin. Measure-
ments
and
morphological assessments were made
with
a
calibrated ocular micrometer
at a
magnification
of
X400.
Length, head shape, tail shape,
and
body shape
were
recorded
for 40
mff/sample.
Head
shape
was
con-
sidered tapered
if the
width decreased upon successive
measurements anteriorly (Fig.
1); if the
sides remained
parallel,
the
head
was
categorized
as
blunt (Fig.
2).
Although many posterior ends were curved, only those
that
had a
distinct hook
at the tip
were labeled
as
such
(Fig.
1,4).
Data were analyzed using
SAS
(SAS Institute Inc.,
1989). Comparisons
of
total lengths
of the
microfilar-
iae
were made between panthers
and
bobcats
and be-
tween morphological types using ANOVA
with
a
split-
plot design where
felid
type
was the
whole-plot
factor
and
morphological type
was the
sub-plot factor. Three
morphological types (tapered
head
and
straight
tail,
blunt
head
and
straight
tail,
and
tapered head
and
but-
ton-hooked tail) were included
in
this
analysis. Micro-
filariae
with
blunt head
and
button-hooked
tails
were
not
included
due to low
prevalence
(1
found
in
pan-
thers
and 3
found
in
bobcats). Confidence limits were
not
calculated
for
percentage
of
prevalence
of mff in
panthers because
the
number examined represented
nearly
all of the
extant population
of
panthers, thus
rendering confidence
limit
calculations
moot.
Results
and
Discussion
Thirty-five
(74.5%)
of 47
adult
(>2 yr of
age)
Florida
panthers
sampled
from
1986
to
1993
were
mff+
at
some
point
in
their
lives
and all
except
for 2,
which
had low
counts
of 20 and
330
mff/ml,
remained
positive
on
subsequent
tests.
Because
panthers
were
usually
sampled
at
2-yr
intervals,
the
average
age
when
animals
be-
come
mff+
is not
precisely
known.
None
of the
5
panthers
tested
at 6 mo of age
were
mff+,
2
of 10
(20%)
tested
positive
in the
1-yr-old
class,
and
15 of 23
(65%)
of
panthers
were
test
posi-
tive
at 2-4 yr of
age.
Of the 23
panthers
that
were
10
yr of age or
older,
22
(96%)
were
mff+.
Counts
ranged
from
10 to
7,380
mff/ml
of
whole
blood.
Microfilarial
counts
showed
fluc-
tuations
with
no
apparent
trends
when
compar-
isons
were
made
between
animals
or
over
peri-
odic
sampling
of
individual
panthers.
Adminis-
tration
of
ivermectin
at the
time
of
capture
seemed
to
have
no
effect
on the
long-term
mi-
crofilarial
intensities.
Because
the
panther-cap-
ture
interval
was
normally
every
2 yr, it was
Copyright © 2011, The Helminthological Society of WashingtonCopyright © 2011, The Helminthological Society of Washington
LAMM
ET
AL.—MICROFILARIAE
IN THE
FLORIDA PANTHER
139
Figure
1.
Microfilaria
from panther blood with tapered
head
and
hooked
tail. Total length
= 332
u.m.
Figure
2.
Microfilaria from panther
blood
with blunt head
and
straight tail. Total length
= 319
jxm.
Figure
3.
Microfilaria from panther
blood
with tapered
head
and
straight tail. Total length
= 329
m.
Figure
4.
Microfilaria from
panther
blood
with blunt head
and
hooked
tail. Total length
= 323
u.m.
impossible
to
assess
the
short-term
effect
of
this head, straight tail (TS);
and
tapered head, but-
anthelmintic
on
microfilaremias
in the
Florida ton-hooked tail
(TH).
Microfilariae
with tapered
panther. heads
and
straight tails were
the
most prevalent
Panther
microfilariae
were
of 4
morphological
(72.1%),
followed
by
those with blunt heads
and
types (Fig. 1-4): blunt head, straight tail (BS); straight tails (21.8%).
The
next most common
blunt
head, button-hooked tail (BH); tapered were
mff
with tapered heads
and
button-hooked
Copyright © 2011, The Helminthological Society of WashingtonCopyright © 2011, The Helminthological Society of Washington
140
JOURNAL
OF THE
HELMINTHOLOGICAL SOCIETY
OF
WASHINGTON, 64(1),
JAN
1997
itu
J\.JUIM>/AI^
*ur
i
no
ncLivuiN
i
rujLAjiju^rtL
OUV^IE,
i i
v^r
wrtonn-svj
IUIN,
otvi;,
J/\IN
iyy
I
tails (5.7%)
and,
lastly, those with blunt heads
and
button-hooked tails (0.4%). Body shapes
were
not
used
as
criteria
for
distinguishing
filar-
ial
types because shape appeared
to be a
highly
variable parameter.
The
average lengths
of the mff
from
panthers
measured
in
fresh blood samples
(n
=
280)
was
320
/mi
(273-370
/mi).
The
widths ranged from
4 to 5
/mi.
All of the mff
measured remained
in
their
original whole blood sample
for a
period
of
2-8 hr
before analysis. Courtney
and
Garber
(1983)
found,
in
measurement
of D.
immitis
and
D.
reconditum
mff
collected from dogs
and
stored
in
EDTA
for 8 hr,
that there
was not a
significant
change
in
microfilarial
width
or
length.
In an
earlier study, Sawyer
et
al.
(1963)
determined that
the
width
of D.
reconditum
stored
in
blood
for 4-6
hours increased
an av-
erage
of 0.5
/mi
above
the
average
of
those mea-
sured
immediately. Both authors concluded that
there
was no
change
in
morphological features
during
this time.
Because
bobcats
and
panthers inhabit
the
same habitat
and D.
striata
has
been reported
in
the
bobcat,
a
screening
of 3
bobcat blood sam-
ples
and
comparison
of mff
between
the 2
spe-
cies
was
performed.
Of the 3
bobcats examined,
all
had
high
microfilaremias.
The
average length
of
bobcat
mff
measured
(n =
120)
was 333
/mi
(297-363
/mi)
and the
average width
was 5
/mi
(4-6
/mi).
A
majority
of the mff
(72.5%)
had
tapered heads
and
straight tails,
20% had
blunt
heads
and
straight tails,
5% had
tapered heads
and
hooked tails,
and
2.5%
had
blunt heads
and
button-hooked tails. This distribution
of
mor-
phologic types
is
similar
to
that
of the
panther
(see Table
1).
Neither
felid
type, morphological
type,
nor the
interaction between felid type
and
morphological type
had a
significant
effect
on
microfilarial length
(P =
0.3375,
0.3572,
and
0.4178, respectively).
Knott's procedure
was the
chosen method
for
studying
morphological features because
it
caused less deformation
and
better delineation
of
the
worms than
did the
Difil
test. Jackson
(1977)
noted that
the filtration
step
of the
Difil
test
procedure seemed
to
cause shrinkage
in
length
as
compared
to
those measurements
ob-
tained from
a
Knott's preparation
and
that
the
filter
membrane would often trap
the mff
mid-
way
through
and
obscure observation
of the
tail.
The
Difil
method
was
used
to
obtain microfilar-
ial
counts because
it was
faster
and was
deemed
Table
1.
Distribution
and
average
lengths
of
Flor-
ida
panther
and
Bobcat
microfilariae
based
on
morphology.
Head/tail
N
Tapered/straight
Panther
202
Bobcat
87
Blunt/straight
Panther
6
1
Bobcat
24
Tapered/button-hooked
Panther
16
Bobcat
6
Blunt/button-hooked
Panther
1
Bobcat
3
%
72.14%
72.5%
21.79%
20.0%
5.71%
5.0%
0.35%
2.5%
X
319
333
323
326
320
338
320
339
SD
7.16
6.16
8.25
6.14
8.52
6.17
0
7.02
Range
(M-m)
276-368
297-363
274-366
299-350
290-368
311-350
320
322-354
a
more sensitive detection method
by
House
and
Glover (1974),
who
reported that
the
Difil
test
is
97.5% accurate whereas
the
Knott's procedure
is
only
89%
accurate.
From review
of
necropsy data
and the
mor-
phological data presented here,
it can be
inferred
that
D.
striata
is the
only species
of filariid
pres-
ent in the
Florida panther. Comparison
of the
microfilarial
length values with those published
for
various
Dirofilaria
spp.
shows that they
are
most similar
to D.
striata,
although references
regarding measurements
of
this
filariid in
felids
are few and
reported microfilarial lengths
vary
significantly
depending
on
processing methods.
Anderson
and
Diaz
(1959)
isolated unsheathed
mff
from
the
uterus
of
adults with
a
length
of
235-270
/mi
and a
width
of 5
/mi.
The
mea-
surements
of
formalin-fixed
D.
striata
from bob-
cats
reported
by
Orihel
and Ash
(1964)
averaged
348
/mi
(327-371
/mi)
long
by 4-5
/mi
wide,
and
those
measured from hematoxylin-stained
thick blood
films
were
230-240
/mi.
Redington
et al.
(1977)
reported
finding
similar dimensions
though
no
values were given.
The finding of
such
a
high prevalence
of mff in
adult Florida
panthers warrants
definitive
microfilarial identi-
fication as
well
as
continued screening
of
whole
blood samples
and
investigation into
the
subtle
health effects that such high numbers
of
circu-
lating
mff may
pose.
Acknowledgments
The
authors thank
N. G.
Keeling,
A. J. An-
derson,
R.
Ball,
R.
T
McBride,
D. S.
Maehr,
E.
Copyright © 2011, The Helminthological Society of WashingtonCopyright © 2011, The Helminthological Society of Washington
LAMM
ET
AL.—MICROFILARIAE
IN THE
FLORIDA PANTHER
141
D.
Land,
J. C.
Roof,
J. W.
McCown,
D. K.
Jan-
sen,
O. L.
Bass,
V. L.
Gibaldi,
T. S.
Ruth,
and
S. H.
Parker
for
their cooperation
in
collection
and/or preparation
of
biological samples
and M.
Dunbar
for
supplying
the
1993 blood samples
and
for
access
to
panther medical records. Grate-
ful
acknowledgment goes
to D. J.
Forrester,
C.
H.
Courtney,
and M. G.
Spalding
for
their expert
advice
in the
editing
of
this manuscript.
We
wish
to
extend
a
special acknowledgment
of and
ded-
ication
to C. M.
Glass
for
assistance with data
collection
and
laboratory analysis
as
well
as ex-
pert advice
and
support. This paper
is
published
as
Florida Agricultural Experiment Stations
Journal Series
No.
R-05017.
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Copyright © 2011, The Helminthological Society of WashingtonCopyright © 2011, The Helminthological Society of Washington