A Survey for the Microfilariae of the Canine Heartworm, Dirofilaria immitis, in the Calgary Region of Southern Alberta


Frimeth, J.P.; Arai, H.P.

Canadian Veterinary Journal 25(1): 14-16

1984


A blood survey for the prevalence of the microfilariae of the canine heartworm, Dirofilaria immitis was conducted in the Calgary area of southern Alberta between November 1977 and August 1979. A total of 514 blood samples was examined by the modified Knott's test. All of the samples were negative for D. immitis microfilariae. Wright's stained blood smears taken from 19 animals at the Calgary Zoo also proved negative. One smear from a male two-toed sloth (Choloepus didactylus) contained sheathless microfilariae which were distinguishable from those of D. immitis. These results, as well as mail survey data indicate that D. immitis is not endemic in the Calgary area. It is recommended that the modified Knott's test be used for similar large scale sampling studies. In addition, it is suggested that the testing of both native and exotic zoo animals which could serve as definitive hosts of D. immitis be continued. These animals may become local sources of infection or introduce other species of microfilariae which will have to be differentiated from those of D. immitis.

A
Survey
for
the
Microfilariae
of
the
Canine
Heartworm,
Dirofilaria
immitis,
in
the
Calgary
Region
of
Southern
Alberta
J.P.
FRIMETH
AND
H.P.
ARAI
Department
of
Biology,
University
of
Calgary, Calgary,
Alberta
T2
N
I
N4
SUMMARY
A
blood
survey
for
the
prevalence
of
the
microfilariae
of
the
canine
heart
-
worm,
Dirofilaria
immitis
was
con-
ducted
in
the
Calgary
area
of
southern
Alberta
between
November
1977
and
August
1979.
A
total
of
514
blood
samples
was
examined
by
the
modified
Knott's
test.
All
of
the
samples
were
negative
for
D.
immitis
microfilariae.
Wright's
stained
blood
smears
taken
from
19
animals
at
the
Calgary
Zoo
also
proved
negative.
One
smear
from
a
male
two
-toed
sloth
(Choloepus
didactylus)
contained
sheathless
microfilariae
which
were
distingui-
shable
from
those
of
D.
immitis.
These
results,
as
well
as
mail
survey
data
indicate
that
D.
immitis
is
not
endemic
in
the
Calgary
area.
It
is
recommended
that
the
modified
Knott's
test
be
used
for
similar
large
scale
sampling
studies.
In
addition,
it
is
suggested
that
the
testing
of
both
native
and
exotic
zoo
animals
which
could
serve
as
definitive
hosts
of
D.
immitis
be
continued.
These
animals
may
become
local
sources
of
infection
or
introduce
other
species
of
microfi-
lariae
which
will
have
to
be
differen-
tiated
from
those
of
D.
immitis.
RESUME
La
recherche
de
larves
du
filaire
Diro-
filaria
immitis,
dans
le
sang
de
chiens
de
la
region
de
Calgary,
au
sud
de
l'Alberta
Cette
etude
dura
de
novembre
1977
jusqu'en
aoat
1979
et
elle
consistait
rechercher
la
presence
de
larves
du
filaire
Dirofilaria
immitis,
dans
le
sang
de
chiens
de
la
region
de
Calgary,
au
sud
de
l'Alberta.
Les
auteurs
exami-
nerent
514
echantillons
de
sang,
a
l'aide
de l'epreuve
modifiee
de
Knott,
et
ils
obtinrent
des
resultats
negatifs.
Des
frottis,
prepares
avec
du
sang
de
19
animaux
du
jardin
zoologique
de
Calgary
et
colores
selon
la
methode
de
Wright,
donnerent
egalement
des
resultats
negatifs.
Un
echantillon
san-
guin
preleve
chez
un
paresseux
a
deux
doigts
male,
Choloepus
didactylus,
contenait
des
larves
de
filaires,
mais
elles
differaient
de
celles
de
D.
immitis.
Les
resultats
de
cette
etude
et
d'un
-
questionnaire
postal,
indiquent
que
la
dirofilariose
canine
n'est
pas
enzoo-
tique
dans
la
region
de
Calgary.
Les
auteurs
recommandent
l'utilisa-
tion
de l'epreuve de
Knott,
pour
des
etudes
d'envergure,
et
la
continuation
de
l'examen
du
sang
des
animaux
locaux
et
des
animaux
exotiques
de
zoo,
susceptibles
de
servir
d'hotes
definitifs
de
D.
immitis.
Ces
animaux
pourraient
devenir
une
source
locale
d'infection
ou
introduire
d'autres
es-
peces
de
filaires
qu'il
faudrait
differen-
cier
de
D.
immitis.
INTRODUCTION
Canine
dirofilariasis
is
a
progressive,
insidious
disease
(9)
which
has
been
reported
from
a
variety
of
mammals
including
man
(22).
It
is
worldwide
in
distribution
being
primarily
asso-
ciated
with
tropical
and
subtropical
zones,
but
also
occurs
in
temperate
areas
(21).
The
problem
of
dirofilaria-
sis
in
Canada
has
attracted
more
atten-
tion
in
recent
years,
as
indicated
by
the
number
of
reports
(2,6,8,25,26,27,28,
29,30)
and
the
numbers
of
dogs
rou-
tinely
examined
(29,30).
The
presence
of
the
etiological
agent
Dirofilaria
immitis,
has
been
reported
from
every
province
except
Prince
Edward
Island.
Results
from
nationwide
"Heart
-
worm
in
Canada"
mail
surveys
have
revealed
an
increased
confidence
in
the
reliability
of
blood
testing
and
clinical
diagnosis
of
canine
dirofilariasis
(25,26,27,28,29,30).
It
is
generally
accepted
that
the
blood
concentration
techniques
are
the
most
accurate
and
preferred
methods
for
the
examina-
tion
of
blood
for
D.
immitis
microfila-
riae,
while
the
smear
technique
is
the
least
accurate
and
preferred.
The
validity
of
clinical
diagnosis
of
heart
-
worm,
is
questionable
unless
em-
ployed
in
conjunction
with
another
diagnostic
technique
such
as
a
blood
concentration
test.
Dogs
infected
with
D.
immitis
may
exhibit
a
variety
of
generalized
clinical
signs
that
may
be
confused
with
other
diseases
(3)
and
pathological
changes
associated
with
heartworm
disease
may
occur
well
in
advance
of
the
clini-
cal
signs.
As
many
as
25
adult
D.
immitis
may
be
present
in
the
absence
of
any
clinical
signs
and
as
many
as
61
worms
may
be
found
in
the
definitive
host
before
the
disease
becomes
clini-
cally
obvious
(23).
It
is
unlikely
that
such
heavily
infected
animals
would
be
native
to
Calgary.
Quantitative
heart
-
worm
surveys
report
mean
intensities
of
most
naturally
infected
dogs
to
be
between
one
and
ten
worms,
even
in
areas
where
the
disease
is
highly
pre-
valent
(5,13,31,32).
Ultimately,
demonstration
of
the
microfilariae
of
D.
immitis
in
the
peri-
pheral
circulation
of
an
infected
animal
is
necessary
for
the
confirma-
tion
of
the
disease.
Blood
testing
reveals
the
presence
of
the
microfila-
riae
of
D.
immitis
in
most
cases,
with
the
exception
of
single
sex,
occult,
or
not
yet
patent
infections.
Immunolog-
ical
tests
such
as
the
ELISA
(10)
and
indirect
fluorescent
antibody
(IFA)
(34)
will
reveal
these
types
of
infec-
tions.
However,
these
tests
are
imprac-
tical
for
large
scale
testing
due
to
the
cost
and
time
involved
in
their
applica-
tion.
The
modified
Knott's
test
(14)
is
Present
address
of
Dr.
Frimeth:
Department
of
Biology,
University
of
New
Brunswick,
Fredericton,
New
Brunswick
E3B
6E1.
Reprint
requests
to
Dr.
Arai.
14
Can
Vet
J
1984;
25:
14-16.
the
recommended
procedure
for
large
scale
sampling
because
of
its
accuracy,
cost
and
time
involved
for
its
use
(20).
Although
canine
dirofilariasis
has
been
reported
from
the
Calgary
area
(25,26,27,28)
comprehensive
surveys
have
not
been
conducted
to
determine
the
prevalence
of
D.
immitis.
A
recent
study
has
demonstrated
that
some
mosquito
species
present
in
the
Cal-
gary
area
are
capable
of
supporting
development
of
D.
immitis
to
the
third
stage
under
laboratory
conditions
and
these
larvae
are
infective
to
dogs
(8).
Although
the
climatic
conditions
of
Calgary
suggest
that
natural
trans-
mission
of
the
nematode
can
occur
(7)
the
absence
of
a
critical
number
of
infected
animals
may
be
the
most
sig-
nificant
limiting
factor.
However,
the
large
immigration
of
dogs
to
the
Cal-
gary
area
from
southern
Ontario
and
parts
of
the
United
States,
where
heartworm
is
endemic,
as
well
as
the
movements
of
local
dogs
could
pro-
vide
this
critical
number.
The
objective
of
this
study
was
to
conduct
a
comprehensive
survey
of
the
blood
of
canines
in
the
Calgary
area
to
determine
the
prevalence
of
the
micro-
filariae
of
D.
immitis.
A
similar
study
of
some
native
and
exotic
mammals
housed
at
the
city
of
Calgary
zoo
was
also
initiated.
Dirofilaria
immitis
has
been
reported
from
a
variety
of
mam-
mals
and
it
was
expected
that
this
sampling
could
reveal
local
sources
of
infection
other
than
in
dogs.
MATERIALS
AND
METHODS
During
the
period,
November
1977
to
August
1979,
blood
samples
were
obtained
from
dogs
from
the
Calgary
Humane
Society,
the
University
of
Calgary
farm
(via
the
Calgary
Animal
Shelter)
and
through
the
cooperation
of
private
owners
and
veterinarians.
Results
from
blood
samples,
breed,
sex,
age,
weight,
hair
length
(classified
as
"long"
or
"short")
and
residential
area
of
the
dog
were
recorded.
Wher-
ever
possible,
the
travel
history
of
the
animal
was
determined
in
the
event
that
it
may
have
been
to
an
area
endemic
for
D.
immitis.
This
informa-
tion
was
imperative
for
"show"
or
"field
-trial"
dogs
attending
national
or
international
competitions.
Blood
was
sampled
irregularly
throughout
the
day
and
evening,
according
to
standard
methods
(11,16). A
1
to
3
mL
sample
was
taken
from
either
the
saphenous
or
cephalic
vein
of
each
animal
and
transferred
to
a
5
mL
EDTA
Vacutainer.
1
All
of
the
samples
were
held
at
room
tempera-
ture
and
examined
by
the
modified
Knotts'
test
within
24
hours.
In
all
cases,
the
entire
stained
pellet
was
examined
microscopically
at
100X
magnification.
Blood
samples
from
zoo
animals
(Table
I)
were
taken
at
intervals
by
zoo
personnel,
smears
prepared,
stained
with
Wright's
stain
and
examined
at
100X
and
400X
for
microfilariae.
RESULTS
Blood
samples
from
514
dogs
and
20
zoo
mammals
were
examined
for
the
presence
of
D.
immitis
microfilariae.
No
evidence
of
a
microfilaremia
was
detected
in
any
of
these
animals
with
the
exception
of
a
male
two
-toed
sloth.
Blood
samples
from
this
animal
con-
tained
sheathless
microfilariae
which
were
distinguished
from
those
of
D.
immitis
by
the
smaller
size,
and
arrange-
ment
of
the
nuclei.
It
was
not
possible
to
identify
these
microfilariae
to
spe-
cies.
However,
they
most
closely
re-
sembled
microfilariae
belonging
to
the
genus
Dipetalonema.
DISCUSSION
Despite
the
fact
that
microfilariae
of
D.
immitis
were
not
recovered
during
the
course
of
this
study,
reports
of
the
presence
of
infected
dogs
in
the
Cal-
gary
area
necessitated
an
investiga-
tion,
even
though
these
infections
were
known
to
be
acquired
elsewhere
(25,26,27).
Since
the
completion
of
this
study,
at
least
one
heartworm
infection
was
diagnosed
in
a
dog
which
had
not
previously
left
the
province
(30).
There
are
certain
inherent
difficul-
ties
in
evaluating
the
prevalence
of
D.
immitis
using
data
obtained
from
blood
sampling
for
the
microfilariae.
Among
these
are
time
for
sampling,
low
microfilaremia,
method
of
exami-
nation,
sample
size,
presence
of
imma-
ture
worms,
single
sex
infections
(1),
and
the
phenomenon
of
occult
filaria-
sis
(33).
Duration
of
the
sampling
period
and
the
number
of
dogs
sampled
should
have
alleviated
some
of
these
problems.
Although
some
blood
sam-
ples
were
taken
during
the
winter
months
when
microfilaremias
are
low-
est
(17),
these
represented
a
small
per-
centage
of
the
total
sample.
The
irregu-
latory
of
sampling
hours
should
not
have
been
significant
as
the
microfila-
riae
of
D.
immitis
are
not periodic
(4).
The
modified
Knott's
test,
employed
in
this
study
could
not
have
eliminated
the
problem
of
single
sex,
occult
or
not
yet
patent
infections.
Employing
sero-
diagnostic
techniques,
such
as
the
ELISA
and
IFA
tests
concurrently
may
have
enhanced
this
study.
How-
ever,
their
availability
at
the
time
of
this
study,
costs,
and
the
time
involved
in
their
application,
prevented
their
use.
Similar
blood
surveys
have
not
sampled
dogs
less
than
one
year
old
(15,18,24).
Although
some
of
the
dogs
sampled
in
this
study
were
less
than
this
age,
their
numbers
were
not
signif-
icant.
In
an
earlier
study
(12)
18
of
635
TABLE
I
SPECIES
AND
NUMBERS
OF
ZOO
MAMMALS
EXAMINED
FOR
DIROFILARIA
IMMITIS
MICROFILARIAE
Species
Number
examined
Lemur
(Lemur
catta)
Macaque
(Maraca
fuscata)
Gorilla
(Gorilla
gorilla)
Two
-toed
sloth
(Choloepus
didactl•lus)
Arctic
fox
(Alopex
lagopus)
Polar
bear
(Thalarctos
maritimus)
Swan
Hills
grizzly
bear
(Ursus
horribilis)
Spectacled
bear
(Tremaretos
ornatus)
Cheetah
(Acionyx
jubatus)
Cougar
(Fells
concolor)
Ocelot
(F.
pardalis)
Jaguar
(Panthera
onca)
2
2
4
2
3
'Becton
Dickinson
Canada
Inc.,
Mississauga,
Ontario.
15
dogs
between
six
months
and
one
year
of
age
were
found
to
be
infected
with
D.
immitis.
In
addition,
to
the
possibil-
ity
of
these
young
dogs
harboring
adult
worms,
a
microfilaremia
may
also
be
detected
should
transplacental
transmission
of
the
microfilariae
occur
(19).
Although
none
of
the
blood
samples
of
animals
tested
from
the
Calgary
Zoo
showed
the
presence
of
D.
immitis
microfilariae,
the
regular
monitoring
of
zoo
animals
is
recommended.
Many
of
these
animals
are
known
to
be
sus-
ceptible
to
heartworm
disease
or
orig-
inate
in
areas
from
which
D.
immitis
is
endemic.
Also,
these
animals
may
introduce
other
species
of microfila-
riae
which
will
have
to
be
differen-
tiated
from
those
of
D.
immitis,
as
demonstrated
in
this
study.
ACKNOWLEDGMENTS
Primary
financial
support
was
pro-
vided
by
grants
from
the
National
Science
and
Engineering
Research
Council
to
the
junior
author,
while
additional
funding
was
obtained
from
the
Alberta
Kennel
Club.
We
wish
to
thank
Pat
Ovenden
who
provided
use-
ful
comments
and
Irene
Johnston
who
typed
the
final
manuscript.
REFERENCES
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ALTMAN
NH.
Laboratory
diagnosis
of
Dirofi-
laria
immitis:
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Bradley
RE,
Pacheco
G,
eds.
Canine
heart
-
worm
disease:
The
current
knowledge.
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University
of
Florida
Press,
1972:
87-93.
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TKR,
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Vet
J
1975;
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244-245.
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ria
immitis.
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1976;
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333-346.
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GIBSON
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immitis
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216-228.
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JF.
SLOCOMBE
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filaria
ill7MiaS
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triseriatus
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low
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the
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-
1980.
HC
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V.M.
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FRIMETH
JP.
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immitis.
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of
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184.
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FRIMETH
JP.
ARAI
HP.
Some
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