immunological studies of the host parasite relationship of
TRANSCRIPT
University of Central Florida University of Central Florida
STARS STARS
Retrospective Theses and Dissertations
1975
Immunological Studies of the Host Parasite Relationship of Immunological Studies of the Host Parasite Relationship of
Dirofilaria Immitis in Domestic Canines Dirofilaria Immitis in Domestic Canines
Douglas Felton Qualls University of Central Florida
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STARS Citation STARS Citation Qualls, Douglas Felton, "Immunological Studies of the Host Parasite Relationship of Dirofilaria Immitis in Domestic Canines" (1975). Retrospective Theses and Dissertations. 179. https://stars.library.ucf.edu/rtd/179
•
HlMUNOLOGICAI, STUDIES OF 'fHE HOST PARASITE RELATIONSHIP OF
DIRO FILARIA I MMITIS IN DOMESTIC CANINES .- -
BY
DOUGLJS FEI,TON QUALIS B,S" !"lorida Technological University. 1973
THESIS
Submitted in partial fulfillment of the requirements for the degree of Me.s tel' of Science in the Graduate Studies Program of Florida Technological University
Orlando, Florida 19'15
1"1 Pl ""-) ..
ACKNOWLEDGMENT •
I would like to express my appreciation to each
member of my committee as well as the faculty and
staff of the Department of Biological Sciences,
Florida Technological University, for their valuable
help in completing this study. I would especially
like to thank Dr. Michael J. Sweeney and Mrs. Dellian
Fay Qualls for their understanding and support.
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ABSTRACT
Canine immunoglobulins of the IgG group were shown to
be intimately associated with the cuticle of circulating
non-infection Dirofilari!, immitis microfilaria. The sig
nificance of these immunoglobulins in blocking a complete
immune respons e in the definitive host is discussed.
Irdmunodiffusion, immunoelectrophoresis and fluores
cent inhibition techniques are described and their appli
cability to the study of host-parasite relationships are
evaluated, A rapid reproducible method for the production
of specific~.lly labeled anti-g. i=i tis microfilaria
globulins is presented.
• TABLE OF CONTENTS
ACKNOWLEDGMENT It •••••••••••••••••••••••••••••••••••••
ABSTRACT ••••••••••••••••••••• 10 •••• It It It It • It •• It • It • It It ••• It •
LIST OF TABLES It • • • • • • • • • It It • It It •• It • • It It • It ••• It It • It • • • It ••••
LIST OF FIGURES It • It ..... . It • It ••• It It • It It ••• It It It It ••• It •• It ••• It •
INTRODUCTION • It It It •• It It It • It • It • It •••• It • It • It • It It It It •• , ••• • , • It • It
PHASE I . A Rapid Batch Method for the Production of Specific Fluorescein IsothiocyanateLabeled Globulins to Dirofilaria immitis Microfilaria • It • It •• It ••• It •••• It • It •••• It It It •• It • It • It It • It •
PHASE II. The Significance of Native Canine Protein Interference with Immune Response to Infection with Dirofilaria immitis ••••••••••••••
GENERAL DISCUSSION •• It It • It ••• It It • It t It It It It ••• It •••• It •• It ••• It It
FOOTNOTES • It It It It • It • It •• It • It •• It •• It It It It It It It • It It It • It It It •• It •• It •• It It
BIBI,IOGRAPHY It •• It •••• It • It •• It It ••••••••• It • It It •••• It •• It • It • It •
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8
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22
41
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LIST OF TABLES •
TABLES page
1. Some Potential Vectors of Dirofilaria immitis in which complete larval development has been reported .•••••••••••••• 28
2. Resul ts of Immunodiffusion Studies • ••••••••• 29
J. Serum Samples Collected From the Domestic Canine Population ••.••.•••.•.••..•• 30
If. Rasul ts of Immunoelectrophorosis Studies •••••.•.•.•..........•......•.•...••. :31
•
FIGURES
1.
2.
3.
4.
LIST OF FIGURES
Life Cycle of Dirofilaria immitis • ••••••••••
Distribution of Dirofilaria immitis
Reaction to Diethylcarbamazine Dihydrogen Citrate (D.E.C.) When Administered Repeatedly to Same Dog
• ••••••••
•• •••••••
FloVi Diagram fer Production of Specifically Labeled Globulins ••••••••••••••
5. Example of Immunodiffusion of Antigenic Test Materials .........•...•..•...
6. Interpretive Line Drawings of Immunodiffusion Example Slide •••••••••••••••
7. Example of Immunoelectrophoresis of Antigenic Test Materials .••• •.••....•. _ ••
8. Interpretive Line Drawings of Immunoelectrophoresis Example Slides .................•........ " ...••••••..
9. Flow Diagram for Production of Specific Antisera for Immunologic Test ..............•....•.•...•....•..•.• , •.•
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•
Early studies of Dirofilaria immitis (canine heartworm).
since its description by Joseph Leidy in 1856, were greatly
h indered by a l ack of knowledge about the life cycle of this
filarial parasite . The elucidation of the life cycle by
Thomas In 1929 was possibly the most significant step to
date in understanding the disease proces s , The problems
Thomas faced stemmed from the complexity of the life cycle
which was finally established by Kume in 1950 and is out
lined in F'ig. 1.
Dirof j larie. ilnmi tj s has been demons tra ted in every
part of the globe except Antarctia. although its distri
bution is predominant in areas with substantial mosquito
populations. An abbreviated world distribution is shown
ill Fig, 2 (Kume et al, 1969), Table 1 lists 47 species
of mosquitoes from which third stage, infectious. micro
filaria (MFF) were recovered (Bemrick and Sandholm 1965 ).
The diYersi ty of species with the capability to act as
secondary hosts of D. immitis may account for its world
distribution. however high vector potential for a given
species under natural conditions is dependent uponJ the
survival of the infected mosquitor the population density
of the !JpeciesJ the accessibility of infected canines with
sufficient microfilarial densities in the peripheraJ cir-
cUlation and the frequency of relationships between the
mosquito and the host (Yen 1938 cited by Summers 1943).
The most important species of mosquito in terms of the
above factors is Culex pipens guinguefasciatus (Bemrick
and Sandholm 1965).
The association of D. immitis with Culex ~. may be
significant in the transmission of this filariate to pri-
2
mary hosts other than canines. While D. immi tis is pri- <E:-marily host specific for canines adult nematodes were
demonstrated in domestic felines, muskrats and humans.
The importance of these genera as hosts can not be evalu-
atad hecause of the scarcity of information on these in-~ fecti ons . Less than two dozen infections are documented
in man in the United States, however filarial infection
of various other species are of greater significance in
other parts of the world.
The culmination of the disease in canines is cardiac
insufficiency associated with hepatic failure. The adult
nematodes when present in the heart in sufficient number,
simply block the flow of blood through the heart and in
terfere with the valvular function. An insufficient
blood supply to the liver and other organs results in
premature organ failure and ultimately death. The active
canine between four and six years of age devclopes an
incapability which is influenced by environmental factors.
Al though this loss of activity is of greater significance
in working dogs, the inability of household pets to per
form normal daily play activities may result in stress to
the ' dog owner or his family (Weiner and Bradley 1972).
The treatment for canines infected with adult nema
todes involves the use of arsenical compounds designed
to eliminate the parasite from the host. The resultant
dead nematodes are flushed into the lungs and often cause
pneumonia in the host. If large numbers of adult nema
todes are present this pneumonic condition often results
in the death of the host during treatment, Chemoprophy
lactic treatment with dimethylcarbamazine as a filarcide
may also result in the death of the host (Fig, J) if
adult nematodes are present (Kume et al. 1969), Because
of these risks an early diagnosis of infection when the
adult nematode loads are low or nonexistant is of utmost
importance.
Diagnosis of filariasis is commonly made by the
detection of microfi laria in the blood or tissue of the
infected host (Kagan 196J). In cases with clinical
manifestations the microfilaria are often not demonstra
ble by current methods, thus the Gstablishment of some
immunological test for diagnosis is much desired (Altme,
1972) ,
The diagnosis of human filariasis, with homologous
antigen prepared from adult Wuchereria bancroft\. or
Wuchereria ma lay\. (elephantiasis) is practically impoo-
J
sible (Tanaka et al. 1970). Consequently the cross-re
action of the antigen prepared from heterogenous species
of adult filaria were applied. The antigens used in
these studies were crude extracts of adult filarial
4
worms and microfilaria and they lacked specificity
(Wheeling and Hutchison 1971). While complement fixation,
bentonite flocculation, hemagglutination, latex agglu
tination, and delayed hypersensitivity skin-test were
performed with these antigens, none of these techniques
have been effective in early diagnosis of filarial in
fection in man.
The reactivity of skin-test antigens prepared from
microfilaria as well as adult Dirofilaria immitis were
compared in persons infected with W. bancrofti (Franks
1946). The dermal responses to these antigens were so
varied that reliable diagnosis was not possible.
Seven antigens from D. immitis were evaluated for
their cross-reactivity with Litomosoides carinii (cotton
rats), W. bancrofti, ~. malayi, Dipetalonema witei
(gerbils) and Setaria cervi (cattle) (Fujita et al. 1970).
Cross-reactivity among filarial species was found in all
combinations.
In 1961, Pacheco carried out a study on the speci
ficity and sensitivity of Q. immitis antigen against in
fected dog sera and "immunized" rabbit sera and reported
that crude extracts of adults worms were not specific.
Furthermore, he studied the cross-reactions between Q.
l.mmitis antigen and the sera of animals infected with
other parasites, and found that there existed cross-re
actions with cases of Ascaris lumbricoidies (ascaris),
and Schistosoma §2. (schistosomiasis) (Pacheco 1961).
5
Dogs given intravenous injections of live non-in
fectious D. immitis microfilaria collected from the pe~
ripherial circulation of a donor individual, demonstra
ted the ability to clear microfilaria from their circu
lation promptly after a second course of injections (Wong
196). Serum from these "immunized" dogs also aggluti
nated live D. immitis microfilaria when the two were mixed
at room temperature. The ability to agglutinate micro
filaria was transferable from immunized individuals to
normal canines by serum transfer.
When plotted against time, the results of complement
fixation, microfilaria agglutination and delayed hyper
sensitivity Skin-test of sera from experimentally infected
dogs , form a curve that can be related to stage develop
ment (Fig, 1) of Q. immitis in the host (Pacheco 1966) .
Antibody was first noted when larvae started migration
from the subcutaneous tissues to the heart (stage 2). The
titers increased throughout the migration period (stage 2)
until the time when microfilaria were shed from adult nem
atodes present in the infected individual's heart (stage
5). after which time the antibody titers started to de-
crease, Thus, it would seem that active migration caused
an immune respons e and that microfilaria absorbed anti
body, It is clear, however, that microfilaria of them
selves do not remove all the antibody present because re
sidual titer is obs erved (Franks 1946), Therefore the
question of a change in the antigenic nature of the host
parasite relationship during this period as suggested by
Schofield in Acanthocheilonema perstans infection is sig
nificatn (Schofield 1957),
The inability to demonstrate an antibody titer with
any regularity in individuals with active infections due
to D, i mmitis, leaves the demonstration of microfilaria
in the peri ph erial blood as the diagnostic method of
choice (Altman 1972), The absence of microfilaremia in
many canines with D, immitis has stimulated the further
investigation of immunological reactions, which might
provide diagnostic techniques as well as a better under
standing of the host-parasite relationship, Due to the
complexity of the life cycle of D, immitis (Fig, 1) and
the complicated nature of its relationship with a defin
itive host, a study of the immune response at every phase
of the infection is far too vast for a single work,
The purpose of this study was to examine the immuno
logical processes invoked during the time when micro
filaremia can be demonstrated, This period affords three
advantages to the investigator, first, the acquisition of
6
7
microfilaria can be arranged without euthanasia of the
hosts, second, the supply of microfilaria is virtually un
limited, third, the diversity of the microfilarial stages
found during this period constitutes representative forms
from an important part of the life cycle.
For reasons of clarity this study was separated into
two phases. The first phase was to establish a biological
assay method which would lend itself to the immunological
study of D. immitis microfilaria. A direct fluorescent
antibody technique was chosen for two reasons, first,
because of its usefulness in fluorescent inhibition
studies, second, for its value as a diagnostic tool. The
second phase of this study was designed to evaluate the
complex immune response which appears to take place be
tween the microfilaria and the host. Two immunological
techniques, immunodiffusion and immunoelectrophoresis,
were chosen to evaluate this response. These techniques
were chosen for their ability to separate an apparently
complex immune reaction into a unique series of visible
precipitant bands. While the study was divided into Phase
I and II, evaluations of the interelated techniques of
acquisition and the handling of D. immitis microfilaria
are discussed throughout.
· .
Phase I. A Rapid Batch Method for the Production of Specific Fluorescein Isothiocyanate-Labeled Globulins to Dirofilaria i mmitls Microfilaria.
The recent development of the rapid-batch technique
for labeling antibodies has provided a quick method for
the production of large quantities of fluorescently la-
beled antiserums to various antigens for fluorescent-in-
hibition studies. Where both the quantity and the spec
ificity of the antibody are important considerations,
8
the speed and reproducibility of the rapid-batch technique
are of special value.
The specificity of the antiserum is demonstrated by
the capability of unlabeled antibody to block the immuno
logic reactivlty of the fluorescent antibody. Methods for
acquisition and storage of microfilaria are outlined and
further immunologic test reactions are suggested.
Materials .!ill!!. Methods
Two methods for acquisition of MFF were used. first,
500 ml sample of citrated whole canine blood from an in
fected canine containing D. immitis MFF, as observed by
microscopic examination, was oubjected to a modified sep
aration procedure (Jaffe and Doremus 1970).
The hyperfilaremic blood was hemolyzed with 0.0) M
sAline solution (1 blood. 7 hypotonic saline solution,
9
v/v). The mixture was centrifuged at 1,000 x g for 12
minutes, and the supernate was discarded. The pellet was
suspended in an equal volume of 0.14 M saline solution and
centrifuged at 1,000 x g for 6 minutes. The supernate
containing the white blood cells was removed and discarded,
The MFF and remaining cellular debris were exposed to 10
ml of a 7.5% w/v saponin-water solution for 10 minutes.
Centrifugation at 1,000 x g for 6 minutes removed unde
sired cellular products in the supernate. The micro
filarial pellet was washed sequentially 4 times in 0,14 M
saline solution and centrifuged, Intact MFF free of blood
and cellular debris was observed microscopically, The
microfilaria (2 ml) were emulsified in 2 ml of complete
Freund 's adjuvant (Wong 1963) and stored at _400 C for
later use in antiserum production. Second, whole canine
blood with a high count of MFF Do,ooo to 60,000 MFF/ml)
was collected into heparin or EDTA in vacutainer tubes,
The blood was hemolyzed with equal volumes of distilled
water and centrifuged at 1,000 x g for 10 minutes. The
supernate was discarded, and the process repeated until
microscopic examination showed no traces of blood or cel
lular debris, (Wheeling and Hutchison 1971) and the peJlet
appeared cream-colored (a minimum of 4 waRhings), For
immediate test purposes, the MFF were stored in distilled
water. For long term storage, the MFF were stored at 40 C
in a stor'lge medium (0,017 M NaCl, 0.005 M KCl, 0.002 M
10
MeC12, 0.04 M Na2HP04 , 0.004 M glucose, 100,000 units of
penicillin-streptomycin snd 200 ml of human serum per liter
solution) •
New Zealand white rabbits were immunized with 0.5 ml
portions of the prepared antigenic material, the initial
intravenous injection and then four intraperitoneal in
jections being given at five day intervals between injec
tions. Blood was harvested and serum removed seven days
after the fifth injection by ear laceration, aspiration
and centrifugation. This was stored at 40 C.
A standardized ammonium sulfate precipitation was per
formed on 6 ml of the serum protein (Calcagono et al. 1973).
The vtashed protein pellet was suspended in 3 ml of 0.01 M
phosphate-buffered saline solution (PBSS), and the excess
salts were removed by column chromotography.l Fractions
(6 ml) collected , using 0.13 M PBSS at the flow rate of
1.5 ml/minute. Elution of protein was monitored by mea
surement of absorption at 280 nm on a double-beam spec
trophotomer. 2 Eluates from the highest absorption regions
were concentrated to 3 ml by ultrafiltration (40 C, 50,000
molecular weight membrane, 1.75 kg/sq cm nitrogen).3 The
globulin solution was diluted to 30 ml, and the prot;'ir
concentration determined (absorbance at 280 nm) (Calcagono
et al . 1973). The diluted-globulin solution was added to
3 ml of carbonate-bicarbonate buffer (1 M, pH 9.5) and 10
mg of fluorescein isothiocyanate (FITC)/g of protein.
11
This mixture was stirred for 20 hours at 40 e, It was con
centrated to 6 ml by ultrafiltration as previously de
scribed. The conjugated mixture was separated from excess
FITe carbonated by column chromatography.l High-protein
aliquots were concentrated to 3 ml, using ultrafiltration,
The 3 ml of labeled globulins were added to 15 g of equili
brated (sodium phosphate buffer, 0.01 M, pH 7.5) diethyl
aminoethyl (DEAE) cellulose (5 g DEAE/ml of labeled glob
ulin). This slurry was stirred 10 minutes with a spatula
and centrifuged at 1,500 x g at 40 e, Duplicate sequential
elutions were made on the DEAE-globulin mixture by adding
15 ml volumes (1 ml/g of DEAE) of 0,1 M and 0.2 M PBSS.
The slurry was stirred for 10 minutes at 40 e each time.
All supernates were pooled and centrifuged at 17,000 x g
for 5 minutes at 40 e to remove the DEAE fines. Filtration
through a fine-pore ceramic filter also served to remove
excess DEAE fines, The eluted labeled globulin solution
was concentrated to a final volume equivalent to 1 mg of
protein/ml of solution by ultrafiltration,
For immunologic studies, live D, immitis MFF isolated
by procedures outlined above were washed and suspended in
distilled water for use as antigenic test material. Pr9-
cleaned (70% ethanol) slides were used for preparing heat
fixed, methanol-fixed, and wet-mount antigen slides. One
or two drops of antibody were applied to fixed prepara
tions and these were incubated in a moist chamber for 30
12
minutes at 370 C, The slides were washed twice in 0,13 M
PBSS, dipped twice in dis tilled Vlater, and air-dried.
Wet mounts were prepared from antigen samples which
were incubated in the presence of labeled antibody for 30
minutes at 370 C and subsquently washed by centrifugation
(1,000 x g) in distilled water.
Slides for fluorescent-inhibition (FIH) test Vlere
fixed as outlined in the preceding paragraphs and exposed
to unlabeled antibody for 30 minutes at 370 C in a moist
chamber. The FIH slides Vlere washed repeatedly in 0,13 M
PBSS and distilled water to remove unbound antiserum,
They were reincubated for 30 minutes at 370 C in a moist
chamber in the presence of labeled antibody and then
washed, as previously described, before microscopic exam
ination.
Gel diffusion studies were performed with glass slide
techniques (Ouchterlony 1968). Precoated agar slides Vlere
covered with 2 ml of a 1.5% (w/v) Noble agar solution,
punched in a standard seven-well pattern,4 and tested for
precipitant reactions against anti-D. immitis microfilaria
globulins (MAG), using MFF antigensr whole canine serums,
and human serums,
Immunoelectrophoresis was performed with standardized
laboratory procedures (Ouchterlony 1968). Human anti
human control serums were used to test the techniques.
The MFF antigens, as well as whole canine serum, were test-
13
ed against the MAG.
A simplified flow-diagram of the procedures is given
(Fig. 4) .
Results
'rhe microfilarial isolation procedures. which used the
saline solution and a surfactant (saponin), were time con
suming and yielded agglutinated MFF . The antigenic ma
terials derived from these procedures, although useful for
a~tiserum production, were not utilizable as antigenic
test materials. Procedures which used distilled water atl
a lysing agent for the removal of cellular contamination
from the MFF were rapid and yielded l ive MFF suitable fer
immunologic test purposes .
The rapid-batch method for the production of FITe
labeled globulins was readily applicable to the study of
D. immitis MFF . Large quantities of antiserum wHh high
specificity against the MFF were obtained. Preparations
of MFF incubated in the presence of fluorescent-labeled
MAG were highly fluorescent, showing a +4 on a 0 to +4
scale as compared to standard antisera with high titers .
This fluorescence varied along the cuticle of the MFF .
Although untreated live MFF displayed a slight auto
fluorescence, it was no higher than +1, using the 0 t o
+4 scale .
The specificity of the labeled antibody was demon
strated by the ability of the unlabeled antibodies to
block the fluorescent activity. Specificity was also
evidenced by the ge l diffusion and the immunoelectro
phoresis procedures. In these studies, the reactivity of
~~G toward nonrelated antigenic materials (human and
bovine serums) was absent, whereas the reactivity toward
homologous antigenic components was positive.
Live MFF obtained with the distilled water procedure
were active up to ten days after collection when stored
at 40 C in storage medium.
•
15
Phase III The Significance of Native Canine Protein Interference with Immune Response to Infection with Dirofilaria l.mmi tis.
The specificity of the antibody-antigen reaction has
long been established. The usefullness of this specificity
in immunodiffusion and immunoelectrophoresis studies of
immune response are well documented in the literature.
The results obtained in the first phase of this work reveal
a complex immune reaction which involves antigenic ally
active substances on or in close association with D. immitis
microfilaria.
The antigenic activity observed in the fluorescent
antibody studies, may have arisen from any combination of
the following situations I firstly, microfilarial metabo-
lities in close association with the cuticle, secondly,
native canine protein in intimate association with the
cuticle and thirdly, from the antigenicity of the cuticle
itself.
The purpose of this phase was to evaluate these three
possibilities and to gain additional information on th~
total immune response that takes place under natural con
ditions. Immunodiffusion and immunoelectrophoresis studies
of infected canino serum, normal canine serum, and son-
icated as well as intact microfilaria vlere performed.
16
Materials ~ Methods
The microfilarial pellet obtained as previously de
scribed Vias then suspended in 0.14 M saline solution
(isotonic) for 24 hours at room temperature to remove any
remaining host contaminants (Wheeling and Hutchison 1971).
After washing with distilled water the microfilaria were
suspended in distilled water to give an approximate count
of 30,000 to 60,000 MFF/ml . This suspension was divided
into three equal parts, one-third was emulsified with an
equal volume of complete Freund's adjuvant. The remaining
two-thirds were emulsified with an equal volume of Freund's
incomplete adjuvant. Both suspensions were stored at _40 0
C for later use in antiserum production.
Another sample of whole canine blood containing a high
count of D. immitis MFF were subjected to an alternate sep
aration procedure. The hyperfilaremic blood was centri
fuged at 1,000 x g for 10 minutes. The supernate was col
lected an subjected to additional centrifugation until it
was clear of microfilaria and cellular contamination.
The whole canine serum was separated into three ali
quots. One was emulsified with Freund's complete adjuvant
and the remainder was emulsified with Freund's incomple~e
adjuvant. Both suspensions were stored at _40 0 C for
later use in antiserum production.
New Zealand white rabbits were immunized with 0.5 ml
portions of tho prepared antigenic material . Initial
17
intravenous injections of the Freund's complete adjuvant
suspensions were followed at five day intervals by four
intradermal injections of the Freund's incomplete adjuvant
suspensions. Seven days after the fifth injection blood
was collected by cardiac puncture. Whole blood was placed
in a 370 C incubator for 30 minutes to allow clot for
mation. Clotted blood was moved to a cold box at 40 C for
24 hours to allow clot retraction. Serum was removed after
centrifugation at 1,000 x g for 10 minutes. Antisera to
Q. immitis MFF (MAG) and anti-infected canine serum (DSAG)
were separated into 1 ml aliquots and stored at - 400 C.
Absorption of anti-infected canine serum (DSAG) with
normal canine serum5 was accomplished as follows. A con
stant volume of DSAG (0.1 ml/tube) was added to a series
of ten 5 ml serological test tubes. Normal canine serum
was added to the DSAG to give final volume ratios of III
to 1110. A constant volume of normal canine serum (0.1
ml/tube) was added to a second set of tubes. Anti-in
fected canine serum was added to these tubes to yield a
final ratio of 112 to 1110. Combined sera were incubated
at 370 C for one hour and placed in a cold box at 40 C for
a minimum of 24 hours to allow the antigen antibody co~
plex to form. The absorbed DSAG was stored at -400 C.
Antigenic test material Vias collected as close as
possible to material used in antisera production. Samples
of infected canine serums were collected from the domestic
16
canine population by local veterinarians (Table J). Micro
filaria from previous separation procedures were separated
into two aliquots. One aliquot was stored at _400 C in
tact while the other was sonicated prior to storage. The
sonicators power was set at the maximum. 6 Sonication was
carried out in 15 second intervals in an ice bath until the
microfilaria were completely disrupted as determined by
microscopic observation (Wheeling and Hutchison 1971).
Immunodiffusion was performed by modification of a
standard glass slide technique (Ouchterlony 1966). Pre
cleaned glass microscope slides (25 x 75 mm) were washed
in 1% (v/v) acetic acid solution to remove residual oil.
Acid washed slides were rinsed in distilled water (minimum
of six times). Rinsed slides were dipped in 70% ethanol,
ignited and allowed to burn until the ethanol was removed.
Precleaned slides were coated with a thin coat of 1% (w/v)
agrose and stored at 40 C for later use. Precooled agrose
coated slides were placed into racks and coated with 1.5%
(w/v) Noble agar (4 ml/slide at 560 C) and returned to a
40 C cold box to cure (Palmer and Woods 1972). After cure,
the prepared slides were punched with a five-well pattern4
and the plugs removed by a vacuum with a Pasteur pipettq.
Antisera used for immunodiffusion was diluted in nor-
mal saline in single unit increments from 111 to 1110.
Diluted as well as undiluted solutions of MAG, DSAG and
absorbed DSAG were applied to the center well of prepared
•
19
immunodiffusion slides (Fig, 5),
Solutions of antigenic test material which incorpo
rated the same concentration gradient given above were pre
pared, Diluted and undiluted solutions of sonicated and
intact microfilaria, as well as infected and ' normal J. ~anine
serum were applied to the outer wells (Fig, 5),
The slides were incubated in a moist chamber at 370 C
for one hour and placed in a 40 C cold box to allow pre
cipitant bands to develop, After 24 to 48 hours at 40 C
the slides were stained by a modified thiazine red stain
ing procedure (Palmer and Woods 1972), Racks of slides
were taken directly from the 40 C cold box and washed in
0,03 M phosphate buffer for four to five hours. Washing
was performed in an excess of phosphate buffer agitated
by a magnetic stirrer. Washed slides were completely
immersed in 1% (v/v) acetic acid for 10 minutes to fix
precipitant bands in place. Fixed slides were rinsed in
distilled water and exposed to 0.3% (w/v) thiazine Red R
for 10 minutes. Stained slides were decolorized for 20
minutes in 1% (v/v) acetic acid with complete change of
solution twice during this period. The developed slides
were finally fixed in 1% (v/v) acetic in 1% (v/v) glycP~ol
solution and dried. The dried slides were inspected on
an opaque viewer and precipitant bands recorded (Fig, 6).
Immunoelectrophoresis tests were also performed by a
modification of the standard glass slide technique
20
(Ouchierlony 1968). Precleaned agrose coated slides were
removed from the 40 C cold box, placed in racks and coated
with 1.5% (w/v) Noble agar in Veronal buffer (4 ml/slide
at 560 C). Slides were returned to 40 C to harden (Palmer
and Woods 1972). After the slides hardened they were
punched with a standard two-well, single trough pattern. 7
Prepunched wells were removed by vacuum with a Pasteur
pipette.
Infected canine serum, normal canine serum, sonicated
MFF and intact MFF were applied to wells and racks of slides
were electrophoresised for two hours at 150 v in Veronal
buffer (Palmer and Woods 1972).8
The troughs were removed from electrophoresised slides.
The antisera were applied to each slide (Fig. 7 and 8).
The slides were incubated in a moist chamber at 370 C for
one hour and returned to a 40 C cold box to allow precip
itant bands to develop. After 24 to 48 hours at 40 C the
slides were washed and stained as before. The slides were
dried and precipitant reactions recorded (Fig. 7 and 8).
Commercial antisera to canine IgG and IgM were ac
quired and immunoelectrophoresis and immunodiffusion pro
cedures were repeated with these sera. 9 The precipitRPt
bands which resulted with the use of these antisera were
compared to those obtained with the antisera produced in
this laboratory (Fig. 6, 7 and 8) .
Common controls were used for both immunodiffusion
21
and immunoelectrophoresis. Human, anti-human control sera
were used as positive controls while normal saline was
used as the negative control.
A simplified flow-diagram of the procedure used in the
development of the antisera and their use in immunodif
fusion and immunoelectrophoresis is shown (Fig. 9).
Results
The microfilarial isolation procedures which used dis
tilled water for a lysing agent and for the removal cellu
lar contamination were rapid and resulted in active micro~
filaria suitable for immunological study. Prolonged wash-
ing in normal saline was a useful precaution for the re
moval of contamination not detectable by microscopic exam
ination. The ability of prolonged washing to remove
native canine protein not in intimate association with the
microfilaria,· was demonstrated by the specific reactivity
of microfilaria with anti-IgG and not with antisera pre
pared against all other serum constituents (Fig. 6 and 7),
I mmunization of New Zealand white rabbits with an- ~ tigens emulsified in Freund's adjuvant was successful in
the production of large quantities of specific antisera.
Intradermal injections in which incomplete adjuvant wa~
used resulted in less trauma to individual rabbits than
did the continued use of the complete adjuvant.
The collection of blood by cardiac puncture was con
venient and resulted in greater quantities of blood than
22
ear laceration procedures.
The results of immunodiffusion studies are seen in
Table 2 and Figure 6. All antisera showed their highest
reactivity when undiluted. Different concentrations of
antisera and antigenic test material did not appreciably
move precipitant banda in diffusion patterns of sonicated
and intact microfilaria. The sonicated as well as the in
tact microfilaria did not migrate extensively. therefore
the antigen-antibody complex was formed ei"tiler very close
to, or within the center well. The antisera to infected
canine serum reacted e~ually to all test sera (Table ).
The immunoelectrophoresis results are given in Table
4 and Figures 7 and 8. The migration of materials ap
peared to accelerate under an electrical potential.
The results of immunoelectrophoresis and immunodif
fusion studies in which commercially produced antisera
was used did not vary from those obtained with antisera
produced in this laboratory (Fig. 6, 7 and 8).
General Discuss ion
The immune response to D. immitis in a susceptible
host indeed must be a very complex se~uence of events.
Other investigators have demonstrated numerous antigerdc
components of both adult nematodes and microfilaria
(Wheeling and Hutchison 1971). However, in order to
assess the immune response, in vitro studies must look at
the microfilaria in the same manner as the natural immune
2)
system. For this reason intact microfilaria as determined
by light rnicroscopy were used to produce antisera f or
immunological studies .
The applicability of fluorescent techniques as rapid.
reproducible diagnostjc tools is well established. Their
usefulness in the diagnosis of filariate-type infections
is evidenced by the results of Phase I of this study . The
value of these methods resides not only in the elucidation
of whole microfilaria in the blood. but in demonstration
and subsequent evaluation using inhibition studies of con
stituents in close relationship to the microfilarial
cuticle.
Nairn (1964) states. "Helminth cuticle is non-anti
genic and effectively screens the tissue of the parasite
from the host. Antibody production is limited therefore
and only occurs against the excretions and/or secretions
of the digestive and reproductive tracts of the worms . "
Although the variable fluorescence seen in fluorescent
antibody studies lends support to the statement that D.
immitis cuticle is non-antigenic. the lack of limitation
of fluorescense to the secretory and excretory portals
tends to discount the statement that antibody formation is
limited to excreta.
Absorption studies using DSAG and normal canine sera
were unable to demonstrate the presence of unique consti
tuents solely during infection. Normal canine serum
24
appea red to contain all the antigenically active material
present in infected canine serum. Unique antigenically
active metabolites were found useful in the diagnosis of
other helminth infections (Nairn et al. 1964). The ques
tion of Q. immit is microfilarial metabolites and their
usefullness as a diagnostic aid is still unanswered.
Further studies should be undertaken to extract these
possible antigenically active metabolites from infected
individual's serum, however in absorption studies with
normal canine serum special care must be taken to insure
the quality of serum used. Microfilaria of D. immi tis ? were demonstrated to be transplacental and therefore,
norma l indjviduals should be at least three generations
removed from exposure to any nematode (Mantonani in press).
It is quite possible that the immune response demon-
stra ted in rabbits in this study was not elicited by the
microfilaria th emselves but only by antigenically active
native canine protein carried on their cuticle. Studies
by Wong (1963) and Pacheco (1966) of serotogical changes
in the infected canine, demonstrated the possible uptake
of canine antibody by microfilaria shed into the circu
lation by adult nematodes present in the heart of the i ~
fected individual. Immunoelectrophoresis in this study
demons trated canine IgG tightly bound to the nematode
cuticle.
The s erological changes which were observed in
25
infected individuals by Wong (196)) and Pacheco (1966)
could represent the formation of 7S immunoglobulin capable
of blocking an immune response to these microfilaria, A
protective serum factor was demonstrated in tumor-bearing
animals (Hellstrom et al, 1969),
Lymphocytes from tumor-bearing individuals were in
hibitory to the growth of neoplastic cells from the same
individuals in vitro, Sera from these animals often con
tained 7S i mmunoglobulins which could bind specifically
to the tumor cell and block the lymphocyte effects (Hel~
Istrom 1967), A similar co-existance of cellular immunity
and blocking factor was shown to exist between mouse embryo
cell and lymph node cells of pregnant mice (Hellstrom
1969), After several pregnancies sired by males of an
other strain, offspring became hyporeactive to allografts
of paternal tissue, although their lymphocytes demon
strated specific immunity to paternal antigens in vitro
(Soren cited by Hellstrom 1969), It is interesting that
maternal to fetal transfer of IgG does occur and this may
account for the results seen in pregnant mice (Gitlin et
aI, and Breyere et aI, cited by Hellstrom 1969),
It l.s appa rent from the work of Hellstrom et aI,
(1969) that some neoplastic cells have the ability to
stimulate the production of blocking antibodies, If block
ing antibodi es also function to protect the antigenically
forei gn fetus it is poss ible tha t the fetus itself may
possess some mechanism to elicit the production of these
antibodies.
26
A similar co-existence of cellular immunity and
blocking antibodies might provide an explanation of the
ability of D. immitis microfilaria to survive in the cir
culation of the primary host.
The immunoglobulin found to be intimately associated
with the cuticle of D. immitis microfilaria was of the
IgG group, a 7S immunoglobulin. The immunoglobulin active
in blocking lymphocyte effects in the tumor system was
also of the 7S group,
In serological studies of infected canines, two anti
body titer peaks were observed, one occurred about a month
after infection the other just prior to emergence of
microfilaria from pregnant female nematodes in the heart
(Pacheco 1966),
If it is assumed that blocking antibody was elicited
on the part of antigenically foreign cells in the tumor'
and fetus systems, then it is possible that other organ
isms may have similar antigenic components which could
elicit blocking immunoglobulins, If this is the case the
rise in titer seen by Pacheco (1966) may represent host
response to these components, thereby protecting the MFF
from cellular immunity,
It is therefore suggested that the immune response
observed in the rabbits in this study was directed not
27
toward the microfilaria but against canine IgG present on
the cuticle. This immune response to IgG would account
for the variable flourescence observed in the fluorescent
antibody work as well as the precipitant band seen in
both immunodiffusion and immunoelectrophoresis. Absor
ption studies in which normal serum was used support
this hypothesis since normal as well as infected canine
serum would contain quantities of IgG.
Needless to say the immune response involved in the
host parasite systems is extremely complex and there is
much to learn about this relationship. In the case of ~.
immitis factors which merit further study are macrophage
migration techniques, immunological enhancement studies
and the involvement of protective material in the cuticle
of microfilaria. Continuation of these studies would
hopefully bring about a better understanding of the host
parasite relationship in D. immitis infection and form
a model for the investigation of other disease phenomenon.
•
28
Table 1. Some potential vectol~ of Dirofilaria immi~is in which complete larval development has been reported.
Aedes aegypti albopictus canadensis caspius edgari fifiensis geniculatus gaumensis infirmatus koreicus pandani polynesiensis pseudoscutellaris punctor solicitans taenirorphynchus togoi triseriatus texans
Anopheles algeriensis crucians earlel freeborni
Anopheles hyracanus pseudopictus hyrcanus sinensis maculipennis plumbeus wnctipennis guadrlmaculatus superpictus walkeri
Culex almulirostris plplens pipiens molestus piplens pall ens pipiens guinguefasciatus restuans si tiens tarsalis terri tans tritaeniorphynchus
Mansonia annulata bonneae dives titillans
Mansonia uniformis Psorophora ferox
SAdapted from original Table by Bemrick and Sandholm 1965. The Journal of Parasitology, Vol. 52, No.4, August 1966, p. 762-767.
Table 2. Results of Immunodiffusion Studies.
Antigen
Sonicated Microfilaria Intact Microfilariac Normal Canine Serum Random Se:1J.m Samples
# la 2
aa ga 7a 8a
9a
lOa 11 12
it 15a 16a 17 18 19a
20a 21 22 23 24 25a
268
+ -+
+ + + + + + + + + + + + + + + + + + + + + + + + + +
Antiserab
DSAG
+
+
+ + + + + + + + + + + + + + + + + + + + + + + + + +
Absorbed-DSAG
------------------
aDenotes known positive for MFF in perpherial circUlation.
bSymbolSI - ~ no reactionl + = precipitant band.
cSerum from individuals three generations removed from exposure to any nematode.
29
30
Table 3. Sarum Samples Collected From the Domestic Canine Population.
Sample Ii Knott.s Testd Ageb ~a Haircoata Breedc
1 + 2 M L/H Mixed 2 + ? M s/H Mixed • 3 - J F s/H Mixed 4 - 4 M s/H G. S.
g + 3 F L/H Mixed + g F s/H G. s.
7 + F s/H G. S. 8 + 3 F s/H G, s. 9 + 4 M s/H Dob.
10 - 2 M ? ? 11 + 2 M L/H Mixed 12 ? ? M ? ? 13 + 4 M s/H Mixed 14 ., 1 M t/H Poodle • 15 + 3 M L/H Collie 16 ? 5 M t/H Poodle 17 + 5 M ? ? 18 ? 3 F ? Mixed 19 + 6 F s/H Boxer 20 + 2 F t/H I. S. 21 ? 3 M L/H Husk;y 22 ? 2 ? s/H G. s. ~4 ? 2 F s/H w.
? ? ? ? ? 25 + 4 M s/H G. s. 26 + 4 M s/H Dob.
M = Male! F " asymbols. + c Positive! - ,. Negative! Female! L/H • Long Hair! S/H = Short Hair! ? c Unknown! G. S. = German Shepherd! Dob. " Doberman pinscher! I. S •• Irish Setter! W. c Weimaraner.
bAge expressed in years to nearest birthday.
cBreed determined by major characteristic.
dAs given in Richard E. Bradley (ed .) Canine Heartworm Disease. A Discussion of tho Current Knowledge. Department of Veterinary Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville.
31
Table 4. Results of Immunoelectrophoresis Studies.
Antigen Antiserab
Anti-Dog Anti-Dog Anti-Dog r1Mi DSAG IgM & IgG IgG IgM
Sonicated MFF + + + + -Intact MFF - - - -Normal Can6ne
Serum + + + + + Random Serum
Sa~ples # la + + + + +
2 + + + + +
a + + + + + + + + + +
Sa + + + + + 6a + + + + + 7a + + + + +
8a + + + + + 9a + + + + +
lOa + + + + + 11 + + + + + 12 + + + + + 13a + + + + + 14 + + + + + lSa + + + + + 16 + + + + + 17a + + + + + 18 + + + + + 19a + + + + + 20a + + + + + 21 + + + + + 22 + + + + +
~a + + + + + + + + + +
2Sa + + + + + 26a + + + + +
aDenotes known positive for MFF in perpherial cir-culation.
bSymbola I - = no reactionl + = precipitant band.
cScrum from individuals three generations removed from exposure to any nematode.
Fig. 1. Life Cycle of Dirofilaria immitis. a
Developmental Time Required Length Of Stage Location For DeveloEment Worm Host
Micro1ilaria Can ne Blood Undetermined 300 "'" Primary • (Stage 5) ! ~
10-48 Days I nfective Mosquito 1,000~ Secondary Larva 1 (Stage 1)
1 (Temperature Dependent)
Infective Canine 3-4 Months 1,000 A. Primary Larva I (Stage 2) Submuscular
Membrane, Subcutaneous Tissue, Adipose Tissue, Subserosa & Muscles
+ Immature Heart & 6-7 Months 3.2 cm Primary Heartworms Pulmonary
l(Stage 3) Artery + Male 15.0 cm Primary Mature Heart & 3 Months
Heartworm Pulmonary Female 28 . 0 cm (Stage 4) Artery
aAdapted from original figure by Kume et al. 1969. In Richard E. Bradley (ed.) Canine Heartworm Disease. A discussion of the Current Knowledge . Department of Veterinary Scl o~ce, Institute of Food and Agricultural Sciences, University of Florida, ~aine5ville.
'" N
Fig . 2. Distribution of Dirofilaria immitis. a
•
aAdapted from ori~inal figure by Kume et al . 1969. III Richard E. Bradley (ed.) Canine Heartworm Disease . , Di scussion of the Current Knowledge . Department of Veterinary Science, Institute of Food and Agricultural Sclences, University of Florida, Gainesville.
bShaded area endemic for Q. immttls .
JJ
Fig. 3. Reaction to Diethylcarbamazine Dihydrogen Citrate (D.E.C.) When Administerea Repeatedly to Same Dog (Spitz, Male, J Years Old With ~licrofilaria and Adult Worms) .
Dos age mg/lb
Medication
Days between medications, and condition of the dog after recovery
Time between medication
2.5
subcut. injec.
and occurrence of reaction 47 (minutes)
Symptoms vomit, loss of vitality I< pale
Resul ts recovered before next morning
2.5
per oral
47
nausea, vomit loss of vi tali ty and pale
recovered after 2 hours
0.5
subcut. injec.
20
loss of vitality lay down, feeble pulse and pale
died before next morning
aAdapted from original figure by Kume et al. 1969. In Richard E. Bradley (ed.) Canine Heartworm Disease. A Discussion of the Current Knowledge, Department of Veterinary Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville.
•
•
Fig. 4. Flow Diagram for Production of Specifically Labeled Globulins.
Infected Animal ~ Hyperfilaremic Blood 1 (lysis of blood cells)
Live Clean Microfilaria
35
Antigenic T:st Material Ne! Zealand White Rabbi ts
~Immunologic Test
I Unlabeled Serum
1 (1 1. V •• 4 I. P. injections)
Whole Rabbit Serum
1 (ammonium ppt. )
sulfate
Reactions Serum Proteins
1 (Sephadex Chromatography)
Globulins---Serum Globulins 1 (FITC - - conjugated)
Labeled Serum Globulins
1 (DEAE Batch Chromatography)
~ __________________________ Specifically Labeled , Serum Globulins
•
Fig . 5. Example of Immunodiffusion of Antigenic Test Mat erials.
)6
Absorbed - DSAG _ ULWa Sonicated Microfilaria - URW
Anti-Dog - IgG - CW
Infected Canine Serum - LLW Normal Canine Serum - LaW
Bgymbols I ULW: Upper Left Wall, URW '" Upper Right Well, CW = Center Well, LRW '" Lower Right Well, LLW z Lower Left Well.
•
Fig. 6. Interpretive Line Drawings of Immunodiffusion Example S Ude.
37
Absorbed - DSAG Sonicated Microfilaria
Infected Canine Serum
IgG~~
IgG \.'lr'gG Anti-IgG
Normal Canine Serum
Pig. 7. Example Test Materials.
+
)8
of Immunoelectrophoresis of Antigsnic
-Normal Bovine Serum _ UWa
Anti-Normal Bovine Serum - T
Normal Saline - IJII
Sonicated Microfilaria -UW
Anti-Dog IgG, IgM - T
Normal Canine Serum - LW
Sonicated Microfilaria -UW
Anti-Dog IgM - T
Normal Canine Serum - IJII
Sonicated Microfilaria UW
Anti-Dog IgG - T
Normal Canine Serum - IJII
BsymbolS. UW. Upper Well, T = Trough, IJII = Lower Well.
39
Fig. 8. Intorpretive Line Drawings of Immunoelectrophoresis Example Slides.
Alb. IgM IgG J ---><!-l Normal Bovine Serum - UWa
Anti-Normal Bovine Serum - T
• IgG
e ~ -
=
- -o A
Ib
Normal Saline - LW
Sonicated Microfilaria - UW
Anti-Dog IgG, I~~ - T
Normal Canine Serum - LW
Sonicated Microfilaria - UW
Anti-Dog IgM - T
Normal Canine Serum - LW
Sonicated Microfilaria - UW
Anti-Dog IgG - T
Normal Canine Serum - LW
~yr.bolB I UW = Upper Welll T c Trough, LW - Lower Welll Alb. u Albumin .
40
Fig. 9. Flow Diagram for Production of Specific Antisera for Immunologic Test.
Infected Animal
1 Hyperfilaromic Blood
(Lysis of blood cells)
Live Clean Microfilaria Infected
(24 hours 1 Normal Saline)
Anti era Antigenic Test_-, Production Material
(DilLtion) (Sonication Dilution)
(1,000 x gi 10 minutes
'=T"~ Antisera Production
(DilLtion Absorption)
~-----+Immunologic Test Reactions+--------Y
FOOTNOTES
lsephadex G-25, 25- by J-cm column, Phal~acia Fine Chemicals Inc., Piscataway, N. J.
2perkin-Elmer, Double Beam Spectrophotomer, Coleman 124, Hitachi, Ltd., Tokyo, Japan.
JDiaflo UM-50, Diaflo Ultrafiltration Membranes, Amicon Corporation, Lexington, Ma.
4Immunodiffusion Punch Set 51450, Gelman Instrument Company, Ann Arbor, Michigan.
5Canino serum three generations removed from any Nematode infection. Dawson Research Corporation, Orlando, Fl.
6150 watts Branson Sonifier Cell Disruptor Heat Systems-Ultrasonics, Inc., Melville, N. Y.
7Immunoelectrophoresis Punch Set 51449, Gelman Instrument Company, Ann Arbor, Michigan.
8Deluxe Electrophoresis Chamber, Gelman Instrument Company, Ann Arbor, Michigan .
9Microbiological Associates, Subsidiary of Dynasciences Corporation, Bethesda, Maryland.
41
42
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