Internarionol Journal for Parasitology. Vol. 9. pp. 275-279.

Pergamon Press Ltd. 1979. Printed in Great Britain.

DIROF1hiRIA IMMITIS: CELL-MEDIATED AND HUMORAL
IMMUNE RESPONSES IN EXPERIMENTALLY-INFECTED DOGS
ROBERT B. GRIEVE,*? BRYAN M. GEBHARDT: and RICHARD E. BRADLEY, SR.*
*College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, U.S.A.
and
*Department of Pathology, College of Medicine, University of Florida, Gainesville, FL 32610, U.S.A.
(Received 9 August 1978)
Abstract-GruwE R. B., GEBHARDT B. M. and BRADLEY R. E. 1979. Dirofilariu immitis: Cell-mediated
and humoral immune responses in experimentally-infected dogs. I nternational J ournalfor Parasitology
9: 275-279. Four dogs were experimentally infected with 30 Dirofluriu immitis infective larvae, four
dogs received two such infections and four dogs served as uninfected controls. A partially-purified
D. immitis antigen was used in an indirect hemagglutination assay to determine anti-D. immitis
antibody titers. Anti-D. immifis antibody was first detected in infected dogs 4 weeks after infection.
Titers were highest 2 weeks after the appearance of microfilariae and diminished to low levels thereafter
in the single infection group. Antibody levels in the double infection group decreased similarly but
were demonstrable throughout the study. Antibody titers were significantly higher in the infected dogs,
but there were no differences in titers between single and double infection groups.
The responses of peripheral lymphocytes to phytohemagglutinin P and pokeweed mitogen were
significantly depressed in infected dogs. Peripheral blood lymphocyte transformation could not be
induced with D. immiris antigens. Differences between groups in T-cell function were not demonstrated
by total hemagglutinating antibody or 2-mercaptoethanol labile hemagglutinating antibody following
immunization with sheep erythrocytes.
INDEX KEY WORDS: Parasitic nematodes; Dirofilariu immitis; dog; cell-mediated immunity;
humoral immunity; lymphocyte transformation; hemagglutination; indirect hemagglutination;
immunosuppression.
INTRODUCTION
STUDIES on the kinetics of the canine immune
response to Dirojilaria immitis have been directed
principally at the humoral immune response
(Pacheco, 1966; Tulloch, Pacheco, Casey, Bills,
Davis & Anderson, 1970; Weiner & Bradley, 1972).
Although an antibody response was demonstrable in
each of these studies, the basis for survival of D.
immitis in the dog and the reasons for successful
reinfection of infected and previously infected dogs
remained unclear. Further, in the controlled studies
employing experimental infections, crude, non-
specific antigens were used to assay antibody
responses which may have limited the amount and
value of the information produced. To date, assays
of the canine cell-mediated immune response to D.
immitis have relied on skin testing (Mantovani &
Kagan, 1967); these investigators studied natural
infections with a TCA-soluble, column purified adult
D. immitis antigen which appeared to be specific and
sensitive in their assay. However, interpretation of
such in vivo assays is difficult, because they do not
tPresent address: Department of Preventive Medicine,
New York State College of Veterinary Medicine, Cornell
University, Ithaca, NY 14853, U.S.A.
lend themselves to quantification. Kobayakawa
(1975) reported on the guinea-pig cell-mediated
immune response to an adult D. immitis extract. A
response was confirmed by the migration inhibition
test, lymphocyte transformation test, skin test, and
the skin reaction by passive transfer with sensitized
peritoneal exudate cells. Cytotoxicity of peritoneal
and splenic cells to microfilariae was demonstrated
in vivo and in vitro. This work confirmed the anti-
genicity of the adult D. immitis homogenate, but the
results are difficult to relate to the canine immune
response to a D. immitis infection.
The present investigation was designed to further
elucidate the nature of the cell-mediated and humoral
immune responses to D. immitis in experimentally-
infected dogs. The humoral response was quantitated
by indirect hemagglutination using a semipurified
antigen reported to be genus and species specific
(Mantovani & Kagan, 1967); the cell-mediated
immune response was studied using a semiquantita-
tive in vitro lymphocyte transformation assay.
MATERIALS AND METHODS
Experimental animals. Twelve pedigree beagle dogs, 6
from each of 2 litters, 9-10 weeks of age, were used
(Hazelton-Saunders, Inc., Midlothian, VA, U.S.A.). The
275
276 ROBERT B.GRIEVE, BRYAN M.GEBHARDT~~~ RICHARD E.BRADLEY,SR.
I.J.P. VOL. 9. 1979
dogs were housed in a Rockefeller-type isolation building
and provided with feed and water ad fib. Precautions were
employed to maintain the rooms insect-free and the dogs
helminth-free. Fecal analyses (Whitlock, 1948) of all dogs
were performed periodically to insure a helminth-free
status. Prior to experimentation all dogs were vaccinated
against canine distemper, canine infectious hepatitis and
leptospirosis. The dogs were divided into 3 groups of 4
animals with 1 male and I female of each litter in each
group. One group was an uninfected control group; the
second and third group received single and double
infections of D. immitis, respectively.
Experimerltal infections. The initial D. immitis infection
was administered when the dogs were IO months-of-age
(Week 0). Approximately 500 female black-eye Liverpool
Aedes aegypti fed on D. immitis microfilaremic canine
blood were obtained from the College of Veterinary
Medicine, University of Georgia, Athens, GA, U.S.A.
Fifteen days later, infective larvae were dissected from the
mosquitos in Hanks balanced salt solution (pH 7.2).
Thirty infective larvae were inoculated via syringe and
20 gauge needle subcutaneously in the inguinal region of
each of 8 dogs. Thirty-six weeks after the initial infection
a second inoculation of 30 larvae was administered to 4 of
the infected dogs.
Humoral immune response determinations. Beginning 4
weeks before infection and continuing until 64 weeks
after infection, blood was collected from each dog at I4
day intervals for serum and microfilaria counts (Weiner
& Bradley, 1970). Anti-D. immifis antibodies were
measured in all sera collected using a semipurified antigen
prepared according to Sawada, Takei, Katamine &
Yoshimura (1965) and Mantovani & Kagan (1967). The
indirect hemagglutination assay (IHA), including antigen
titration, was a modification of a widely used technique
(Kagan & Norman, 1976).
Lymphocyte transformation. Lymphocytes were isolated
by a modification of the method of Thilsted & Shifrine
(1977) 43, 45, 49 and SO weeks after the initial infection.
The resultant lymphocyte-rich preparation was washed in
50 ml of RPM&1640 (Gibco, Grand Island, NY, U.S.A.).
Mononuclear cell numbers in each preparation were
determined by total and differential counts; cell viability
assays were performed to insure consistency between
preparations and experiments. Each cell preparation was
then washed with 50 ml of RPMI-1640 and resuspended
to a final concentration of 1.25 X 10fi mononuclear
cells/ml in RPMI-1640 suoulemented with I Y oenicillin-
streptomycin-mycostatin . (Gibco, Grand I%nd, NY,
U.S.A.) and 10% normal canine serum. Cells were
dispensed at a rate of 2.5 x IO5 cells/culture; each treat-
ment level was assayed in quadruplicate cultures.
Phytohemagglutinin P (PHA) (Difco Lab., Detroit, MI,
U.S.A.) was added in a range of 0.02550.2 Id/culture
while Pokeweed Mitogen (PWM) (Gibco, Grand Island.
NY, U.S.A.) and Concanavalin k(Con A) (Miles Lab.;
Inc., Elkhardt, IN, U.S.A.) were added in ranges of
l-10 Id/culture and l-10 ug/culture, respectively. The D.
immitis antigen used was a combination of equal protein
quantities of male and female aqueous-soluble somatic
extracts in phosphate buffered saline (PBS, pH 7.2) and
was used in a range of IO~lOOug protein/culture. In
preliminary experiments, this antigenic preparation
elicited blastogenic responses in splenic and mediastinal
lymph node cells derived from dogs naturally-infected
with D. immitis. Mitogens and antigens were diluted in
RPMI-1640 and dispensed in a 1Oul volume. The
cultures were incubated for 48 h at 37C in 5% CO,;
0.5 uCi of tritiated thvmidine (Radiochemical Centre.
Amersham, England) diluted in RPMI-1640 was added
to each well and incubation was continued for 16-20 h.
Cultures were harvested and counted by a modification of
the method of Strong, Ahmed, Thurman & Sell (1973).
The amount of incorporated thymidine was expressed as
the mean c.p.m. of the 4 replications at each treatment
level.
immunizution with sheep red blood cells. Three ml of a
20% sheep red blood cell (SRBC) suspension in PBS was
administered intravenously to all dogs 40 weeks after the
initial D. immitis infection and again 16 days later to
determine the responsiveness of infected dogs to a
heterologous antigen. Sera were collected on the day of
primary immunization and 8, 16, 20, 22, 27 and 29 days
thereafter. Total hemagglutinating antibody and 2-
mercaptoethanol (2-ME) labile hemagglutinating anti-
body were measured by a modification of a previously
described technique (Scott & Gershon, 1970). Separate
experiments were performed to insure that the IgM
fraction of canine sera was 2-ME labile.
I nterpretation of data. Because evaluation of lympho-
cyte transformation data revealed there were no statisti-
cally significant differences between experiments, data
from the 4 experiments were combined for statistical
analysis. Differences in mean c.p.m. and antibody titers
between groups were evaluated by analysis of variance.
RESULTS
Microfilariae were first detected 26 weeks after
infection in 1 dog and were present in all infected
dogs by 30 weeks after infection. Mean maximum
microfilariae numbers ranged from 7100 to 36,300
microfilariae/ml and there were no differences
in microfilariae numbers in single and double infected
dogs at any time. One dog that received 2 D. immitis
infections became amicrofilaremic 22 weeks after the
second infection; microfilariae were not detected in
that dog again throughout the study.
Thirty milligrams of the semipurified antigen
preparation was obtained from the initial 120 mg
of TCA-soluble preparation. A dilution of 90 pg of
protein/ml was optimal in sensitizing SRBC with
antigen.
Anti-D. immitis antibody was first detected in
infected dogs 4 weeks after the initial infection
(Fig. 1). Antibody levels in the infected groups were
significantly higher (PC 0.05) than in the uninfected
group. The antibody titers in the single infection
group began to decrease shortly after patency (Week
30) while antibody levels in the double infection
group persisted at low levels through Week 64.
There were no significant differences between
antibody titers of single and double infection groups
after administration of the second infection.
Peripheral blood lymphocyte transformation
could not be induced in D. immitis infected dogs
with any level of D. immitis antigen tested (Table I).
Responses to all mitogens were diminished in
infected dogs, but due to individual variability and
I.J.P. VOL. 9. 1979
D. immitis: immune responses in dogs
277
h
:: 4.0
_, Uninfected t
.e
i i
--~~~~-. Double infection
z
i j
3.5
-** Single infection
.$
. .
.$ 3O
Q 2.5
.L
g
_;j
2.0
::
ti 15
'ij
E
';s I.0
N
$
0.5
g
zz
Weeks after initial D. immifis infection
FIG. 1. Anti-D. immitis antibody responses of dogs receiving single and double D. immitis infections
( f = second infection administered to double infection group).
TABLE~-MITO~ENA~~ANTIG~N INDUCED PERIPHERAL ~L~DLYMPHOCYTETRANSFORMATIONOFDOGS INFECTED W:TH
Diroflariu immitis
Culture Noninfected Single infection
treatment c.p.m. (+ S.&M.) S.i.i C.p.tTl. (+S.E.M.) s.i.
-- --__~ -
None 84 (f6) - 82 (+6) -
0.025 ul PHA 2152 (+756) 25.6 991 (+ 276) 12.1
0.05 PHA 4676 (+ 1363) 556 2192 (+699) 26.7
0.10 PHA 7103 (k 1538) 84.5 *3559 (rt 883) 43.4
0.20 PHAS 8607 ( f 5638) 102.4 3543 (i. 615) 43.2
1 ul PWM 3650 ( f 524) 43.4 **2022 (& 333) 24.4
5 PWM 4163 (k588) 49.5 **2276 (+301) 27.7
10 PWM 3731 (+ 525) 44.4 ***1942 (-+247) 236
1 ug ConAs 5961 (+ 5132) 7@9 2688 ( t 192) 32.7
5 ConA 8501 (+479l) 101.2 4552 (I 889) 55.1
10 ConA 9454 ( f 5585) 112.5 3930 (+ 638) 47.9
10 DIAT ug 96 (+9) 1.1 79 (&5) 0.9
50 DIA 118 (+16f 1.4 87 (i4) 1.0
100 DIA 103 (k 12) 1.2 79 (k4) 0.9
*Statistically significant difference (PC 0.05).
**Statistically significant difference (P<O.O25).
***Statistically significant difference (P<O~Ol).
tStimulation index (c.p.m. stimulated cuIture/c.p.m. unstimulated culture).
*This level was evaluated in one experiment.
This mitogen was evaluated in 2 dogs in each group in 1 experiment.
1 DiroJ laria immitis antigen.
Double infection
C.p.lTl. (kS.E.M.) s.i.
98 (kll) -
1530 (+617) 15.6
2983 (& 1037) 30.4
*3921 ( j: 1145) 40.0
5608 f&2310) 57.2
* *2255 ( + 320) 23.0
**2458 (+ 296) 25.0
***2323 (&244) 23.7
2815 (21786) 28.7
5629 ( rf: 2424) 57.4
4728 (rf: 2192) 48.2
102 (rt9) 1.0
122 (+ 11) 1.2
121 (ItlO) 1.2
sample size, statistical significance was demonstrable responsiveness, characterization of the responses of
only at one level of PHA (PC 0.05) and all levels of infected and noninfected dogs to a T-dependent
PWM (PcO.01). The decreased mitogen responsive- antigen, SRBC, was attempted to further elucidate
ness observed in infected dogs was consistent in each the nature of the immune depression. However,
experiment. there were no significant differences in total or
Since a diminished mitogen responsiveness could 2-ME labile anti-SRBC antibody levels between
be an indication of a generalized depressed immune infected and noninfected dogs.
278
ROBERT B. GRIEVE, BRYAN M. GEBHARDT and RICHARD E. BRADLEY, SR.
I.J.P. VOL. 9. 1979
DISCUSSION
Results obtained on antibody titers were largely
comparable to data from similar studies (Pacheco,
1966; Weiner & Bradley, 1972). Anti-D. immitis
antibody was detectable between 2 and 4 weeks after
infection and peak titers were reached 2 weeks after
patency in all dogs. Since the titers diminish 2-4
weeks after patency, microfilaremia may be associ-
ated with a decrease in circulating antibody. Results
illustrated in Fig. 1 suggest that there was a delayed
anamnestic response in the group receiving 2
infections, but that response was not statistically
significant. Re-evaluation of the data indicated that
this apparent secondary response was related to
unusually high titers in the dog that became amicro-
filaremic. This animal developed high antibody
titers at week 50, just prior to the amicrofilaremic
state and at the same point of the apparent anamnes-
tic response. Therefore, an elevated antibody titer
may be related to establishment of the amicro-
filaremic state.
Mantovani & Kagan (1967) reported that the
TCA soluble antigen, as used in the present investiga-
tion, was both genus- and species-specific based on
IHA and skin testing of naturally-infected dogs. It is
impossible to assess the specificity of the purified,
TCA soluble antigen without testing it against sera
from dogs or other animals harboring other para-
sites, but our evidence indicates that it is more
specific than the crude preparations typically used.
For instance, when using a crude saline-soluble
antigen, Weiner & Bradley (1972) observed that
IHA titers for noninfected dogs were as high as
1 : 256; interpretation of changes in antibody
levels was thus rendered difficult. Our use of a
semipurified antigen enhanced specificity as evidenced
by anti-D. immitis antibody titers of noninfected
animals which never exceeded 1 : 2. Further, the
antigen used during this study may be more sensitive
than crude preparations. Relative antibody titer
increases were greater than titers reported after a
similar infection schedule using a crude antigen
preparation (Weiner & Bradley, 1972). Although
blastogenic activity of the D. immitis antigen
preparation was confirmed in separate lymphocyte
transformation experiments of splenic and medias-
tinal lymph node cells of naturally-infected dogs,
evidence for peripheral blood lymphocyte transfor-
mation could not be obtained. In addition to the
antigens used in the 4 lymphocyte transformation
experiments, the TCA soluble IHA antigen and an
aqueous-soluble somatic microfilaria extract did not
stimulate lymphocyte transformation in small-scale
experiments. In contrast, successful lymphocyte
transformation using similar antigen preparations
has been reported in other filarial infections (Ottesen,
Weller & Heck, 1977; Portaro, Britton & Ash, 1977;
Weller, 1978). Ottesen et al. (1977) used antigens
from saline extracted D. immitis and Brugia malayi
adults to successfully transform peripheral blood
lymphocytes from humans infected with Wuchereria
bancrofii. Portaro et al. (1977) have used
saline extracts of D. immitis, Trichnella spiralis
and Brugia pahatfgi to transform splenocytes for B.
pahangi-infected jirds. Weller (1978) demonstrated a
stage-specific lymphocyte response to larval, adult
and microfilarial antigens in B. pahangi-infected rats.
The failure to induce D. immitis-specific lympho-
cyte transformation in this study may have been
related to the markedly diminished mitogen respon-
siveness in infected dogs. Recently, various types of
immune suppression have been documented for 3
other filarid parasites (Dalesandro & Klei, 1976;
Ottesen et al., 1977; Portaro, Britton & Ash, 1976;
Weller, 1978). Dalesandro & Klei (1976) using
Dipetalonema viteae showed decreased antibody
responses to bovine serum albumin in hamsters and
jirds and to SRBC in hamsters. Immunizations at
different times in the course of infection indicated
that the onset of immunodepression was related to
the appearance of microfilariae. In our study, the
decline in anti-D. immitis antibody titers would
support the idea of a microfilaria-associated immune
suppression. Ottesen et a/. (1977) reported that a
specific cellular unresponsiveness to W. bancrofti
occurred in infected humans. Humans with infec-
tions, humans without detectable infections but
previously exposed to the parasite and humans with
no exposure to the parasite were immunized with
heterologous antigens. Lymphocyte responses to
those antigens, filarial antigens and mitogens
revealed that only the response to the filarial
antigens was impaired in infected humans. In other
work, splenocytes from B. pahangi-infected jirds
were shown to transform with exposure to filarial
antigens, but mitogen responsiveness was inversely
correlated to the appearance of filarial-specific
splenocytes (Portaro et al., 1976). Weller (1978)
demonstrated a hyporesponsiveness to mitogens in
splenic, lymphnode and peripheral lymphocyte
preparations from B. pakangi-infected rats, and
noted rats with responsive node cells averaged less
microfilariae/ml than rats with unresponsive cells.
Presently, assays of T-cells in dogs are limited to
experiments measuring function. Direct erythrocyte
rosettes, previously reported as effective in T-cell
quantitation for the dog (Bowles, White & Lucas,
1975) are not reliable (Krakowka & Guyot, 1977).
We observed that immunization with SRBC
generated no significant differences in total or 2-ME
labile hemagglutinating antibody. Although total
antibody titers were virtually identical between
groups, antibody responses were primarily in the
IgM class of immunoglobulin after secondary
immunization of infected dogs and in non-IgM
classes in noninfected dogs. This observation would
support the hypothesis of impaired T-cell function,
but it could not be substantiated with statistical
analysis.
l.J.P. VOL. 9. 1979 D. immitis: immune responses in dogs 279
The implications of a state of diminished immune
responsiveness in D. immitis infected dogs are
numerous. This phenomenon must be considered in
evaluating the pathogenesis of D. immitis infections.
It is possible that this condition may predispose an
infected dog to infectious and neoplastic diseases.
Further, although preliminary experiments on
immunizing dogs with irradiated larvae have shown
promise (Wong, Guest & Lavoipierre, 1974), the
results of the present investigation demonstrate that
the complexity of the immune response to D. immitis
should be more clearly defined before immuno-
prophylactic regimens are considered.
Acknowledgements-The authors gratefully acknowledge
the assistance of Mr. Joe DiCarlo, Mr. Nguyen Van Dat
and Dr. H. Neil Becker. The D. immitis infective larvae
were supplied by the US-Japan Cooperative Medical
Science Program-NIAID. Published as Florida Agricul-
tural Experiment Station Journal Series No. 1295.
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