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Cell-mediated and humoral immune responses to Dirofilaria immitis in experimentally-infected dogs

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Title:
Cell-mediated and humoral immune responses to Dirofilaria immitis in experimentally-infected dogs
Creator:
Grieve, Robert Burton, 1951-
Copyright Date:
1978
Language:
English
Physical Description:
x, 99 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Antigens ( jstor )
Canines ( jstor )
Dogs ( jstor )
Electrophoresis ( jstor )
Fractions ( jstor )
Gels ( jstor )
Infections ( jstor )
Lymphocytes ( jstor )
Microfilariae ( jstor )
Animal Science thesis Ph. D
Canine heartworm disease -- Immunological aspects ( lcsh )
Dissertations, Academic -- Animal Science -- UF
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 92-98.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Robert Burton Grieve.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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03908473 ( OCLC )
AAB5061 ( NOTIS )

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CELL-MEDIATED AND HUMORAL IMMUNE RESPONSES
TO Dirofilaria irrnitis IN EXPERIMENTALLY-INFECTED DOGS















By
Robert Burton Grieve


A Dissertation Presented to the Graduate Council of
the University of Florida
in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy


University of Florida
1978














ACKNOWLEDGEMENTS

I extend my gratitude to Dr. Richard Bradley, supervisory chairman,

for supporting this research and for the countless lessons he taught.

The suggestions and guidance of the supervisory committee, Drs. P. T.

Cardeilhac, H. L. Cromroy,and E. M. Hoffmann, are appreciated. Dr. M. D.

Young participated in each committee meeting and offered help and

suggestions whenever needed.

The generosity and guidance of Dr. John Neilson is gratefully

acknowledged. I was especially fortunate to benefit from his experience

and special abilities in helminth antigen isolation and characterization.

The insight obtained with his help will be invaluable in future research

and was principally responsible for obtaining an ideal postdoctoral

position.

Dr. Bryan Gebhardt helped in so many ways they cannot all be listed.

He provided boundless time, patience,and expertise in all the aspects

of the immune response researched in this dissertation. He frequently

offered helpful criticism and suggestions and further, he provided a

good deal of encouragement at times when it helped. His assistance is

greatly appreciated.

There have been valued associations within and outside the labora-

tory with fellow graduate students. Dann Brown, Nguyen Van Dat, Carlos

Costa, John Kennedy, Dan Murfin,and Wallace Randell have been remarkable

friends whose special and diverse interests in parasitology contributed

to an interesting and beneficial research atmosphere. Each of them








offered time and suggestions essential to the completion of this work.

Mr. Dat deserves additional thanks for all his help on the statistical

analyses in this investigation.

The assistance of Dr. H. Neil Becker in providing veterinary

services critical to various aspects of this research is appreciated.

Special thanks are due Mr. Joe DiCarlo who provided outstanding

technical assistance in Dr. Gebhardt's laboratory. The assistance

of Mr. Louis Ergle in coordinating technical help in Dr. Bradley's

laboratory is appreciated.

The patience and typing skill of Ms. Dianna Jackson is sincerely

appreciated. This dissertation would have been much slower developing

without her kind help.

The support of all my family is sincerely acknowledged. It is

not possible to describe the many ways they have all helped. However,

a very special acknowledgement is in order for Jane, my wife. She

left a career she enjoyed to move to Florida and has worked at three

mediocre jobs in a town where student wives are both underappreciated

and underpaid. Jane somehow managed a budget and household with very

meager funds, accepted the long, late hours at the laboratory, encouraged

me through the many rigors of graduate study, and somehow found time

to develop several independent interests. She has my deepest respect

and appreciation for all these things and more.

A graduate assistantship was provided by the College of Veterinary

Medicine (Institute of Food and Agricultural Sciences Animal Research

Facility). The research was supported in part by Hatch Project 1419

(W-102).















TABLE OF CONTENTS


Page
. ii


ACKNOWLEDGEMENTS . . . .......

LIST OF TABLES . . . . . . . .


LIST OF FIGURES . . .

ABSTRACT . . . . . . .


. . . vii


. viii


INTRODUCTION .. .....


LITERATURE REVIEW ....


MATERIALS AND METHODS . .

Experimental Animals . . .
Experimental Design . . .
Experimental Infections . .
Rlnnrd rnll rtinns


. . 12

.. . . 12
... . . 12
. . . . . . . 13
1 3


Antigen Purification and Characterization . . . .
Protein Determinations . . . . . .
Crude Antigen Extraction . . . . . .
Preparative Isoelectric Focusing . . . . . .
Production of Rabbit Antisera . . . . . .
Immunodiffusion . . . . .
Polyacrylamide Gel Electrophoresis in Sodium Dodecyl
Sulfate . . . . .
Polyacrylamide Gel Electrophoresis .. . ......
Humoral Immune Response Determinations ....
Purification of the Indirect Hemagglutination Antigen
Indirect Hemagglutination Assay .....
Cell-Mediated Immune Response Determinations ..
Lymphocyte Isolation . . . . . . . . .
Lymphocyte Transformation ......
Lymphocyte Rosette Assay . . . . . . . .
Immune Response to Sheep Red Blood Cells .. ....


14
14
14
16
17


RESULTS . . . . . . . .. . ... 34
Microfilariae Counts ..... .. ... .... ...... 34
Antigen Purification and Characterization .. . ... 34
Preparative Isoelectric Focusing . . ... .... 34
Immunodiffusion .. . .. . .... ... .. 37
Polyacrylamide Gel Electrophoresis in Sodium Dodecyl
Sulfate . . . . . . . . 37


. . . . . .







Page
Polyacrylamide Gel Electrophoresis .. . . . 47
Humoral Immune Response.. .. . . .. ... .... 47
Cell-Mediated Immune Response .. ... .... 50
Lymphocyte Transformation . ... . ...... . 50
Lymphocyte Rosettes.. . . . ........ . 50
Immune Response to Sheep Red Blood Cells . . . 50
DISCUSSION . . . . . . ....... . . 60
Antigen Purification and Characterization . . . .. 60
Humoral Immune Response . . . . . .. . 63
Cell-Mediated Immune Response . . . . . . 65
CONCLUSIONS . . ... ....... .. . . . 71
APPENDICES . . . . .
I. Diroj'iaria innitis Microfilariae Counts Over the
Experimental Period . .. . . .. . ... 74
II. Polyacrylamide Gel Electrophoresis in Sodium Dodecyl
Sulfate: Crude Antigen Preparations and Soluble
Somatic Fractions . . . . . . . ... 77
III. Standard Molecular Weight Curve for 7.5%
Polyacrylamide Gel . . ......... . 79
IV. Standard Molecular Weight Curve for 10%
Polyacrylamide Gel ............... 81
V. Standard Molecular Weight Curve for 12%
Polyacrylamide Gel . ... ........ . 83
VI. Polyacrylamide Gel Electrophoresis: Crude Antigen
Preparations and Soluble Somatic Fractions . .. 85
VII. Anti-Di.vit. Zaia immitis Antibody Titers from All
Dogs Over the Experimental Period . . ... 86
VIII. Mean Anti-Sheep Red Blood Cell Antibody Titers
Before and After 2-mercaptoethanol Treatment . 89
IX. Abbreviations Defined and Used in the Text .... 90
BIBLIOGRAPHY . . . . . . . . .. . ... . 92
BIOGRAPHICAL SKETCH . . ............... 99








LIST OF FABLES


Table Page

i. DPirhfit'nia imitis Male Soluble Somatic Antigen
Fractions Purified By Preparative Isoelectric
Focusing and Characterized By Immunodiffusion,
Polyacrylamide Gel Electrophoresis and Polyacry-
lamide Gel Electrophoresis in Sodium Dodecyl
Sulfate ....... . . . . .... 44

2. Divnfi,,la\ia itnmi.tis Female Soluble Somatic Antigen
Fractions Purified By Preparative Isoelectric
Focusing and Characterized By Immunodiffusion,
Polyacrylamide Gel Electrophoresis, and Polyacry-
lamide Gel Electrophoresis in Sodium Dodecyl
Sulfate . . . . . . . ... .. .. . 45

3. Dirofitaria innitis Microfilaria Soluble Somatic
Antigen Fractions Purified By Preparative
Isoelectric Focusing and Characterized By
Immunodiffusion, Polyacrylamide Gel Electrophoresis,
and Polyacrylamide Gel Electrophoresis in Sodium
Dodecyl Sulfate . . . . . . ..... 46

4. Mean Counts Per Minute and Stimulation Indices From
Four Canine Peripheral Blood Lymphocyte Transformation
Experiments of Dirofi lria i/rintis-Infected Dogs and
Noninfected Dogs . . ... . . .. . 51

5. Percentage of Canine Peripheral Lymphocytes Forming
Nonimmune Rosettes with Human Erythrocytes . . 52














LIST OF FIGURES


Figure Page


1. Diagrammatic Representation of the Scheme Used in
Purification and Characterization of Dirofilaria
initic Antigens .... . . . . . . .24

2. Diagrammatic Representation of the Procedure for
Purification of the Dirofilaria immitis Indirect
Hemagglutination Antigen .... . . . . . 27

3. Mean Numbers (X103 ) of Diaofi arta immitis Microfilariae
Per ml of Blood in Group B (single infection) and
Group C (double infection) . . . . . 36

4. Preparative Isoelectric Focusing Separation and Percentage
of Recovered Protein in the Final Fractions from the
Dirofiucaia irinitis Male Soluble Somatic Extract . . 39

5. Preparative Isoelectric Focusing Separation and Percentage
of Recovered Protein in the Final Fractions from the
Diroj'iaria irnmiti Female Soluble Somatic Extract . 41

6. Preparative Isoelectric Focusing Separation and Percentage
of Recovered Protein in the Final Fractions from the
DLirofilaria immi-tis Microfilaria Soluble Somatic Extract 43

7. Mean log2 of the Reciprocal Anti-Dio.fiZai.a irruiitis Anti-
body Titers in Groups A, B and C Over the Course of
Infection . . . . . . . .... . . . 49

8. Mean log of the Anti-Sheep Red Blood Cell Antibody Titers
in Group A anc' Groups B + C . . . . ... . 54

9. Mean Difference of log2 of Reciprocal Anti-Sheep Red Blood
Cell Antibody Titers Before and After Serum Treatment with
2-mercaptoethanol .................. 56

10. Separation Profile of Pooled Canine Anti-Sheep Red Blood
Cell Serum on Sepharacyl S-200 and the Pools Collected for
2-mercaptoethanol Lability Determinations . . . 59














Abstract of Dissertation Presented to the Graduate Council of
the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy


CELL-MEDIAIED AND HUMORAL IMMUNE RESPONSES
TO Dirofilaria immirtis IN EXPERIMENTALLY-INFECTED DOGS

By
Robert Burton Grieve

March 1978

Chairman: Richard E. Bradley
Major Department: Animal Science

The canine immune response to Dirofilaria inmitis was investigated

in 3 aspects. Antigens from D. inmitis adults and microfilariae were

isolated and partially characterized and the humoral and cell-mediated

immune responses to D. i.mmitis were studied in experimentally-infected

dogs.

Antigens from soluble somatic extracts of D. iiynitis males, females,

and microfilariae were separated using preparative isoelectric focusing.

The fractions obtained by isoelectric focusing were characterized by

polyacrylamide gel electrophoresis in sodium dodecyl sulfate (PAGE-SDS)

and by immunodiffusion against canine sera from D. imr itis-infected dogs

and homologous rabbit antisera. Adjacent fractions from each preparation

were combined on the basis of isoelectric point, similarity of protein

constituents, and antigenic identity. The combined fractions were further

characterized by polyacrylamide gel electrophoresis (PAGE). Gels were

stained with coomassie blue for protein constituents and periodic acid








Schiff's stain and alcian blue were used to detect carbohydrate

moieties. Triton X-100 was used to solubilize cuticle-bound antigens

from D. inmitis males and females; the resultant preparations were

characterized by the same scheme employed for the soluble somatic

fractions.

Soluble somatic preparations of males, females,and microfilariae

were divided into 7, 7,and 3 final fractions, respectively. Considerable

complexity and a wide molecular weight range of constituent proteins

were observed in most fractions. All male and female fractions and

2 of 3 microfilaria fractions reacted with canine sera from ll. immitis-

infected dogs and homologous rabbit antisera. In contrast, the Triton

X-100 solubilized preparations were less complex and demonstrated weak

reactivity against canine sera from D. ir'nitis-infected dogs and rabbit

antisera. Both stains for carbohydrate determinations after PAGE were

unsuccessful with the soluble somatic fractions.

A trichloroacetic acid-soluble, column-purified D. intnitit; antigen

was used in an indirect hemagglutination assay to determine anti-D. inrnitis

antibody titers in experimentally-infected dogs. Antibody titers were

determined in 4 dogs with a single D. imiiti. infection, 4 dogs with a

second D. irn-iLis infection administered after the maturation of the

first infection,and in 4 uninfected dogs. Sera for antibody determi-

nations were collected every 14 days beginning 4 weeks before infection

until 64 weeks after infection. Anti-D. iimmitic antibody was first

detected in infected dogs 4 weeks after infection. Titers were highest

at week 32, 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 diminished as in the

single infection group, but were demonstrable throughout the study.

Antibody titers were higher (p<0.05) in infected dogs, but there

were no significant differences in antibody titers between single

and double infection groups.

The cell-mediated immune response was investigated using peripheral

blood lymphocyte transformation. Lymphocyte transformation could not

be induced with D. ii itis antigens and the response to the mitogens

phytohemagglutinin P, pokeweed mitogen,and concanavalin A were

significantly depressed (p<0.0001) in infected dogs. Efforts to

quantitate thymus dependent lymphocytes (T-cells) using nonimmune T-cell

rosette foundation with human red blood cells revealed no differences

in T-cell numbers in infected and noninfected dogs. All dogs (infected

and noninfected) were immunized with a T-cell dependent antigen, sheep

red blood cells (SRBC), to investigate possible differences in T-cell

functions in infected and noninfected dogs. No differences between

groups in anti-SRBC antibody levels or 2-mercaptoethanol labile anti-

SRBC antibody levels could be demonstrated.














INTRODUCTION

The canine heartwonr, DiroJfiaria innitis, has been the subject of

considerable research. A more complete understanding of this infection

is important both from the standpoint of the severity of the disease it

may cause and possible ways to control the growing incidence of the

parasite (Otto, 1974a). Further, the parasite often serves as a model

for human filariasis research and may offer insights into the control

of those infections. Research on methods to control D. irrenilis has been

substantial but has been directed mainly to chemoprophylaxis and chemo-

therapy. Although chemoprophylaxis is reliable, it is not totally

effective (Grieve and Bradley, unpublished information) and can be

costly and inconvenient. Treatment to kill adult D. immitis is hazardous

because of the danger of drug toxicity and the potential damage of

emboli from dead parasites associated with the arsenical compound

currently employed (Carlisle et al., 1974).

As an alternative to chemotherapy, research on biologically-derived

antigens to be used as vaccines has been emphasized in recent years,

but it has become evident that before effective immunologic methods for

the prevention or diagnosis of such infections can be developed, the

responses of the host to the parasite and the mechanisms by which the

parasite survives in an otherwise immunologically competent, natural

host must be elucidated. The canine immune response to D. irrnitis

has been the topic of several investigations, but these studies have

often relied upon noncontrolled, natural infections or limited animal








numbers. In the few reports of experiments that utilized experimental

infections, crude, nonspecific antigens were used which may have limited

the amount of information produced.

When the data reported from previous investigations on D. ijmitis

immunity are considered, a number of questions become apparent. In an

effort to answer some of the questions, three major objectives were

developed for this dissertation. The three major objectives addressed

in this research were:

1. Isolation and partial characterization of antigens of D. inrnitis

males, females,and microfilariae.

Rationale: Since different life-stages are present in the dog during

the course of infection, information on the actual antigenic constituents

and antigenic relationships of different stages (adult males, adult

females,and microfilariae) is central to understanding the host immune

response. Information on the complexity of the antigenic constituents

of these stages is prerequisite to further study of possible genus-

specific or stage-specific antigens.

2. Elucidation of the humoral immune response in dogs following

primary and secondary experimental infections with D. iinitis.

Rationale: In all previous reports concerning the humoral response

in experimentally-infected dogs a crude, nonspecific antigen was used

(Pacheco, 1966; Weiner and Bradley, 1972). Since a highly specific

and sensitive adult worm antigen has been reported (Mantovani and

Kagan, 1967), it was considered advantageous to use that antigen to

study the antibody response in experimentally-infected dogs.

3. Investigation of the cell-mediated immune response to D. innitis

in experimentally-infected dogs using the in vitro lymphocyte transfor-

mation technique.








Rationale: Assays of the cell mediated immune response to D. inMitis

have been either biological (Mantovani and Kagan, 1967) or have

measured the in vitro response to a D. iinnitis antigen in an unnatural

host (Kobayakawa, 1975). Examination of the response of lymphocytes

from D. irnitits-infected dogs to D. immitis antigens may provide new

information on the nature of the immune response and on possible

mechanisms of parasite survival.

The experimental design of this investigation was intended to help

gain new, fundamental information on the canine immune response to

D. irtnitis infections under controlled conditions. The intent of this

study was to help in better understanding the infection and to explore

the possibilities of any practical applications of that response.















LITERATURE REVIEW

Dirofitaria immitis Leidy, 1856, the canine heartworm, is distrib-

uted throughout the world; it is especially prevalent in warm, coastal

regions and notably rare in Africa (Levine, 1968). In the United States,

the infection appears to be extending from the enzootic Atlantic and

Gulf Coast regions to the north and west and occurs in highly enzootic

rates in certain temperate areas (Otto, 1974a)

The natural habitat of adult D. inwnitis is in the right ventricle

and adjacent vasculature of the dog, Canis familiaris. However, the

parasite has also been recorded as recovered from vomitus, feces, the

eye, brain, spinal cord, abdominal cavity, bronchioles, various arteries,

and abcesses (Otto, 1974b). Wild canines and felines may also serve

as definitive hosts (Otto, 1974b) and a number of naturally-infected

abnormal hosts have been reported including the black bear (Johnson,

1975), beaver (Foil and Orihel, 1975), wolverine (Williams and Dade,

1976), harbor seal (Iledway and Wieland, 1975), California sea lion

(Forrester et at., 1973), domestic horse (Klein and Stoddard, 19771 and

human (Otto, 1974b).

Experimental infections have been used to characterize the

pathogenesis of D. innitis infection in felines (Donahue, 1975). Adult

worm recoveries from experimentally infected cats are low, and dead

worms found in many cats indicate that the dog is a more suitable host.

Macaques have been experimentally infected to study D. ium.mtis infections

of primates (Wong, 1974). Parasites reached maturity only in those








macaques that were chemically immunosuppressed; it was concluded that a

period of 60-90 days of diminished immune responsiveness after infection

is necessary for the worms to reach maturity in the heart.

Adult D. i ruitis live preferentially in the right ventricle and

pulmonary arteries of dogs. Adult males are 120-200 mm long and 700-

900 v wide with a spirally coiled tail characteristic of most filarid

males; females are 250-310 mm long and 1.0-1.3 mm wide (Levine, 1968).

Microfilariae, the first-stage larvae, develop in the uterus of the

adult female worm and are expelled without a sheath, gut, or genital

primordium. The microfilariae are 286-340 v long and 6.1-7.2 w wide with

a tapered anterior end and a straight tail (Lindsey, 1965). Micro-

filariae are present in the peripheral circulation of dogs with mature

infections and have displayed seasonal (Kume, 1974) and daily periodicity

(Pacheco, 1974). The mechanisms for periodicity are unknown (Masuya,

1976) but increased numbers of microfilariae in peripheral circulation

during periods of greater vector availability facilitates perpetuation

of the infection. A relatively constant number of microfilariae remain

in the circulation of any given infected dog. The mechanism for main-

taining a stable microfilariae population is unknown and withdrawal or

addition of large numbers of microfilariae will not alter the population

present in peripheral circulation (Wong, 1964).

Several mosquito species have been identified as proven D. imnrltis

vectors (Levine, 1968). Microfilariae are ingested by the mosquito

at the time of the blood meal and undergo two molts within the mosquito

before they are infective as third-stage larvae. Third-stage larvae

enter the dog where the mosquito has fed and molt in subcutaneous tissue

to fourth-stage larvae in 9-12 days. A subsequent molt to fifth-stage









larvae occurs 60-70 days after infection (Orihel, 1961). A microfilaremia

indicating maturity is usually evident at 6.5 months (Orihel, 1961).

D. impmit.i infections of dogs are typically diagnosed by demonstrating

the characteristic microfilariae in the blood. However, no real corre-

lation can be made between numbers of microfilariae and numbers of adult

worms (Jackson, 1969; Pacheco, 1974). This method of diagnosis is further

complicated by situations where adult worms are present without circu-

lating microfilariae. Single sex infections or infections in an animal

that will respond in a unique immunological fashion to microfilariae

have no circulating microfilariae (Wong et at., 1973).

Serious disease may result from a long-standing infection with

D. wirmtis, especially if a large number of worms are present in the

dog's circulatory system. Adult worms may produce a chronic endocarditis

and dilation of the right heart; lungs may be congested and are typically

effected by the granulomatous responses to thrombi formed from dead

worms (Levine, 1968). Perhaps the most important pathology in the

lungs is the resultant endarteritis and obstructive fibrosis (Adcock,

1961). In chronic cases, the liver may become enlarged and ascites will

ensue. A terminal liver failure syndrome has been described in

experimentally-infected dogs (Sawyer and Weinstein, 1963). Glomerular

changes have also been reported in infected dogs. Although an immune

complex associated etiology has been postulated (Casey et at., 1972),

evidence indicates that lesions are related to damage caused by motile

microfilariae (Klei nL al., 1974; Simpson et at., 1974).

Most reports indicate that there is not a protective immune

response to D. irmnitis. Adult heartworms are patent for at least two








years (Levine, 1968) and microfilariae may persist when transfused into

uninfected dogs for as long as two years (Underwood and Harwood, 1939).

Under natural conditions in enzootic areas, dogs may be repeatedly

infected over a lifetime, but after subjective analysis of such reports,

it appears that the infection levels are not additive.

There is experimental evidence that dogs can be successfully

immunized against D. in[ritis with irradiated D. imnitis infective

larvae (Wong et at., 1974). Dogs that received varying numbers of gamma-

irradiated third-stage larvae injected on different schedules revealed

up to 0% recovery of adult worms after challenge with nonirradiated

larvae. Antibody levels were not high and there was no anamnestic

response after repeated challenge with either irradiated or nonirradiated

larvae.

Since D. iirnmitis is closely related to many of the human filarids

it is often used as a source of antigen in the immunologic diagnosis of

human filarial infections. Cross-reacting antigens among several

filarids have been demonstrated (Neppert, 1974) and the availability

of D. immitis makes it a good antigen source. Early antigenic prepa-

rations for diagnostic use were typically overnight saline extracts of

macerated microfilariae (Franks and Stoll, 1945) or adults (Kagan, 1963).

Presently, saline extracts of lyophilized adult D. immitis are used to

obtain antigen preparations for the serodiagnosis of human filariases

at the National Communicable Disease Center (Kagan and Norman, 1976).

Indirect hemagglutination and bentonite flocculation tests employing

this antigen have been relatively sensitive, but cross-reactivity is

not restricted to filarids. Unfortunately, cross-reactions with the

sera of patients infected with other nematodes, cestodes, trematodes,








protozoans, and even bacteria have been reported (Kagan and Norman, 1976).

Pacheco (1966) reported on the sensitivity and specificity of different

D. in: mis whole worm extracts. In this study, a saline extract of

lyophilized worms, a delipidized saline extract of lyophilized worms,

acid-insoluble and acid-soluble protein extracts,and an ethanol extract

were compared. The acid-insoluble fraction and the ethanol extract did

not react with known positive anti-D. iniitis canine antisera using

indirect hemagglutination. The acid-soluble protein preparation was

the most specific and sensitive of the antigen preparations tested,

but sera of some animals infected with other helminths often cross-

reacted.

Fluorescent antibody techniques have also been used with crude

antigen preparations of adults (Ellsworth and Johnson, 1973; Wong, 1974),

microfilariae (Wong, 1974; Qualls et al., 1975),and third-stage larvae

(Wong, 1974). Wong (1974) reported the ability to discern stage-

specific antibody using an indirect fluorescent antibody technique with

crude adult, microfilarial and larval antigens.

The initial, extensive purification procedures of D. mniretis

antigens were reported in 1965 (Sawada et at., 1965). That work was

designed to isolate filarial skin test antigen to diagnose human

filarial infections. An aqueous, soluble somatic antigen preparation

from adult D. ininitis was treated with trichloroacetic acid and purified

by gel filtration and ion-exchange chromatography. A fraction demon-

strating a good degree of specificity and sensitivity was isolated and

has been proven to be of benefit (Smith, 1971). In later work (Sawada

et aZ., 1970), anion-exchange chromatography, disc electrophoresis,and

isoelectric focusing were employed to show at least 17 different proteins








in the isolated fraction. The various subfractions obtained were variably

reactive in the skin test and were still relatively complex.

The isolation and purification scheme of Sawada et al. (1965) was

repeated to obtain a highly specific and sensitive antigen fraction

for use in the diagnosis of canine heartworm infection (Mantovani and

Kagan, 1967). This antigen was reactive in skin tests and by the indirect

hemagglutination assay it was shown to be specific for D. ivrrnitis when

tested in dogs naturally infected with D. immitis, D. repens, and

Dipetalonema reconditwm. The advantage of the genus and species

specificity of this antigen would be unique but, unfortunately, it has

not been used in studies on the kinetics of the canine humoral immune

response to D. in mitis.

Another study reported that aqueous soluble extracts of D. imnritis

adults and microfilariae were subjected to disc electrophoresis

(Wheeling and Hutchison, 1971). This technique revealed at least 27

and 17 protein bands, respectively. Immunoelectrophoresis of the adult

extract against homologous rabbit antisera showed 10 precipitin arcs;

2 arcs were noted against sera from a patient with suspected filariasis.

One arc developed when the microfilariae extract was subjected to

immunoelectrophoresis against the human sera.

Takahashi and Sato (1976) used a defatted aqueous-soluble extract

of adult D. inrmitis for fractionation. A fraction that was very

reactive and specific in serodiagnosis of Wucherer'ia bancrofti-infected

patients was reported after gel filtration, anion-exchange chromatography,

and ammonium sulfate precipitation. Disc electrophoresis showed at

least five protein bands with activity from the purified fraction.








More recently countercurrent immunoelectrophoresis of D. imnrit-i

male and microfilarial aqueous soluble somatic antigens against homologous

rabbit antisera was used to demonstrate apparent stage-specific antigens

(Desowitz and Una, 1976). Additionally, this same technique was used

with sera from hypermicrofilaremic dogs and rabbit anti-v. irmnitis

antisera to demonstrate circulating soluble D. immitis antigens. Sera

from humans with low microfilaremias or occult infections showed intense

precipitin lines to microfilaria-specific antigens.

The humoral immune responses to experimental D. inmnitis infections

in dogs were studied in long term experiments with experimental

infections by 2 investigators (Pacheco, 1966; Weiner and Bradley, 1972).

In one study, anti-D. imrmitis antibodies were detected by indirect

hemagglutination as early as two weeks after experimental infection, but

could not be detected by either indirect hemagglutination or complement

fixation beyong the ninth month postinfection (Pacheco, 1966). Peaks

of antibody titers were observed at different times over the course

of the prepatent and patent periods. It was postulated that they may

have been related to antigenic changes in the parasite or to different

antibody class responses. The author suggested that the lack of

detectable antibody shortly after patency was due to absorption of most

of the antibody by circulating microfilariae.

Weiner and Bradley (1972) investigated the humoral immune response

after primary and secondary experimental D. immitis infections. A

crude aqueous-soluble adult worm antigen in an indirect hemagglutination

assay was used and serologic responses were similar to those reported

by Pacheco (1966). A peak titer appeared about 22 weeks after infection

and the amount of detectable antibody declined subsequent to the









appearance of microfilariae. Secondary infections administered after

patency of the first infections did not produce an anamnestic response,

but did delay the decrease in antibody titers as had been observed in

the dogs infected only once.

In further work, Weiner and Bradley (1973) used single, radial

immunodiffusion and 2-mercaptoethanol liability to demonstrate that

immunoglobulin class M (IgM) was probably the primary active immuno-

globulin throughout D. innitis infections. They speculated that

immunoglobulin class G (IgG) may also have some role, but it was not

demonstrable by the techniques used.

To date, most assays of the cell-mediated immune response have

relied on skin testing. Mantovani and Kagan (1967) used the skin test

to evaluate the column-purified antigen. The results obtained sup-

ported the serologic observations of D. i nitis specificity.

Kobayakawa (1975) reported on extensive research on the guinea

pig cell-mediated immune response to a defatted adult D. immiitis

extract. A response was confirmed by the migration inhibition test,

the 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. However, in- vitro assays of the canine cell-

mediated immune response to D. inrnitis have not been reported.














MATERIALS AND METHODS

Experimental Animals

Twelve pedigreed beagle dogs aged 9-10 weeks were obtained from

a commercial supplier. Immediately upon receipt the dogs were housed

in a Rockefeller-type isolation building provided with temperature

control and double-screened windows. Water and a commerically available

feed2 were provided ad libitum. Specific procedures were observed to

maintain the rooms insect-free and the dogs helminth-free. The dogs were

from 2 litters with 3 males and 3 females from each litter; throughout

the experiment the animals were divided by sex. Rectal temperatures were

determined daily and fecal analyses (Whitlock, 1948) were performed

weekly on each dog for 12 weeks after arrival. Fecal analyses of all

dogs were performed intermittently throughout the investigation to

insure a helminth-free status. Previous to experimentation all dogs

were subjected to a standard puppyhood vaccination regimen against

canine distemper, canine infectious hepatitis,and leptospirosis.

Experimental Design

The dogs were divided into 3 groups of 4 animals each with 1 male

and 1 female of each litter in each group. One group was an uninfected

control group (Group A), the second (Group B) and third group (Group C)



Hazelton-Saunders, Inc., Midlothian, VA

Gaines Meal, General Foods Corp., White Plains, NY








received single and double D. invn tis infections, respectively.

Experimental Infections

The initial D. inmmitis infection was administered when the dogs

were aged 10 months (Week 0). Approximately 500 female black-eye

Liverpool Aedes aejypti that were fed on D. immibis microfilaremic

canine blood were obtained from the College of Veterinary Medicine,

University of Georgia, Athens.1 Fifteen days after the infected blood

meal the mosquitoes were dissected in Hank's balanced salt solution

(HBSS)(pH 7.2). Infective larvae were individually counted into 1 ml
2
disposable syringes; after 30 larvae were counted into a syringe,

it was filled to 1 ml with HBSS for inoculation. The larval inoculum

was administered subcutaneously in the inguinal region of each dog in

Groups B and C, then the syringes were filled with HBSS and the wash

was inoculated subcutaneously, opposite the initial inoculation. Each

syringe was carefully washed and the wash was examined microscopically

for remaining larvae. Thirty-six weeks after the initial infection

a second infection of the same larval number was administered to

Group C.

Blood Collections

Blood was collected from the cephalic vein of each dog every 14 days

beginning 4 weeks before experimental infection until 64 weeks after

infection. Approximately 10 ml of blood was collected at each sampling;

2 ml were aspirated into a evacuated tube containing EDTA and the



Provided by the U.S.-Japan Cooperative Medical Science Program (NIAID)
2 R
StylexR, Pharmaseal Laboratories, Glendale, CA

3VacutainerR, Becton, Dickinson and Co., Rutherford, NJ








remainder was used for individual serum samples. Serum collected from

whole blood was stored at -350C for anti-D. inmitis antibody determi-

nations. Anticoagulant treated blood was used to count microfilariae

(Weiner and Bradley, 1970) 2 and 4 weeks before infection, 2 weeks

after infection,and then every 14 days beginning 22 weeks after infection.

Antigen Purification and Characterization

Protein Determinations

A standard curve was used to determine protein concentrations of all

antigen preparations. A known quantity of bovine serum albumin was

serially diluted and the absorbance of each dilution was measured at 280

nm using a dual beam spectrophotometer.2 This provided a standard curve

so the absorbance of the various antigen preparations could be related

to a protein concentration.

Crude Antigen Extraction

Adult D. immitis males and females were collected at necropsy from

experimentally-infected dogs. The worms were separated by sex, washed

three times in phosphate buffered saline (PBS)(pH 7.2),and frozen at

-70C. Microfilariae were collected by immersing female D. immitis

recovered from naturally-infected dogs into PBS (pH 7.2) at 4C for 2

hours; the adults were removed and the microfilariae were recovered

after centrifuging3 the PBS at 12,100 X g for 15 minutes at 40C. Micro-

filariae were washed twice in PBS and frozen at -70C.



American Monitor Corp., Indianapolis, IN

Unicam SP1800 Ultraviolet Spectrophotometer, Philips Electronic
Instruments, Mount Vernon, NY
3Beckman J-21C Centrifuge, Beckman Instruments, Inc., Palo Alto, CA








Each whole worm preparation was minced with a razor blade and

mixed with approximately 5 times the worm volume of cold O.01M 2-amino-

2(hydroxymethyl)-l,3-propanediol(tris)(pH 8). The mixture was further

disrupted using a french pressure cell pressI at 2.0 X 104 pounds per

square inch pressure. The homogenate was extracted overnight at 40C on

a continuous rocker2 and then centrifuged at 12,100 X g at 40C for

30 minutes; the supernatant was harvested and the precipitate resuspended

in 10 volumes of 0.01M tris (pH 8) and further extracted for 72 hours.

The combined supernatants served as the crude soluble somatic extract;

the precipitate was extracted with PBS (pH 7.2) until protein was no

longer evident and then subjected to detergent extraction.

Triton X-1003 was used to solubilize any cuticle-associated proteins

by the method of Harris et at. (1971). Only male and female prepa-

rations were in sufficient quantity for this extraction. The protein-

extracted cuticular debris was lyophilized4 overnight and added to 13%

Triton X-100 (pH 7.5) at a rate of 0.1 g/ml and extracted by continuous

agitation at 4C for 72 hours. The mixture was centrifuged at 12,100

X g at 40C for 30 minutes and proteins were obtained by adding 10 volumes

of acetone to the supernatant at -350C. The precipitate was collected

after 24 hours, washed with ether, dried in vacuo and extracted for

24 hours with 0.01M tris (pH 8). The solubilized preparation was dialyzed



AimincoR French Pressure Cell, American Instrument Co., Silver Springs, MD

2Lab-TekR Aliquot Mixer, Ames Lab-Tek, Inc., Westmont, IL

3TritonR X-100, Fisher Scientific Co., Fair Lawn, NJ

4Labconco Freeze Dry-5, Labconco Corp., Kansas City, MO








against distilled water with 0.1% sucrose at 4'C for 48 hours and

lyophilized. The Triton X-100 solubilized fractions of male and

female cuticles are referred to as TSM and TSF, respectively.

Preparative Isoelectric Focusing

Approximately 100 mg of crude male soluble somatic antigen (MSSA),

120 mg of crude female soluble somatic antigen (FSSA) and 80 mg of

crude microfilaria soluble somatic antigen (MFSSA) were individually

subjected to preparative, flatbed isoelectric focusing.1 The method-

ology observed is a modification of the techniques described by Winter

et at., (1975). Each crude preparation was dialyzed overnight against

1% glycine at 4C. A slurry consisted of the aqueous antigen preparation,
2 3
5 g of Sephadex G-75 superfine, 5.0 ml of ampholines pH 3.5-103,and

distilled water to 100 ml was used to prepare each gel bed. The slurry

was mixed, degassed and poured into a glass tray, then evaporated to

reach 75% of the slurry crack-point. The gel bed was subjected to a

continuous electric current at approximately 7 watts of constant

power4 at 10C5 for 14-16 hours. After focusing was complete, the

current was discontinued and a dry filter paper (5 X 25 cm) was placed

over the length of the gel bed for 2 minutes. The paper was removed

and a fractionating grid was pressed through the gel bed. The filter

paper was air dried, washed 3 times for 15 minutes each time in 10%



LKB 2117 Multiphor, LKB Instruments, Inc., Rockville, MD

Pharmacia Fine Chemicals, Pharmacia, Inc., Piscataway, NJ

3AmpholineR pH 3.5-10 LKB Instruments, Inc., Rockville, MD

Electrophoresis Power Supply, ISCO, Lincoln, NB

5Lauda K-4 R/D Brinkman Instruments, Messergate-Werk, Lauda, W. Germany








trichloroacetic acid (TCA), and stained with 0.2% coomassie brilliant

blue1 dissolved in methanol, water, and acetic acid (50:50:10). After

staining was complete the paper was destined with methanol, water, and

acetic acid in the same proportions until the background color was

absent.

The pH of the gel bed at each of the 30 divisions created by the
2
fractionating grid was determined; this data and the staining patterns

of the corresponding filter paper print were used to divide the gel

bed into distinct fractions. When the constituent divisions of each

fraction were determined, the gel bed within those divisions was harvested

and washed into glass columns3 (1 X 30 cm). The columns were eluted

with distilled water and 5 ml volumes were collected until protein was

not detectable. Total protein in each fraction was determined, then the

fractions were dialyzed to remove ampholines, lyophilized,and solubilized

with distilled water to a final protein concentration of 3 mg/ml. If

less than 3 mg of protein was present the fraction was solubilized with

1 ml distilled water.

Production of Rabbit Antisera

Antisera for use in immunodiffusion was made in mature, female

New Zealand white rabbits by intradermal inoculations (Vaitukaitus et

aZ., 1971) of crude soluble somatic or protein-extracted cuticle debris

preparations. One mg of protein of male, female,and microfilarial crude



1Searle Diagnostic, High Wycombe, Bucks-England

2Corning Glass Works, Corning, NY

3Calbiochem, La Jolla, CA









soluble somatic antigens and 1 mg dryweight of male and female cuticular

debris were administered to individual rabbits in a suspension of 1 ml

of 0.01M tris (pH 8) and 1 ml of complete Freund's adjuvant. The

suspension was homogenized by passing it through a 20 gauge micro-
2
emulsifying needle, then inoculated intradennally at 25-30 sites on

the back of each animal. At a separate site each animal received

0.5 ml of a vaccine containing diptheria and tetanus toxoids and a

pertussis immunogen.3 Animals were reimmunized by the same procedure

8 days later using Freund's incomplete adjuvant; the vaccine was not

used at reimniunization. Twenty-nine days after the second immunization

each rabbit received 1 mg of homologous crude antigen by intravenous

administration in a marginal ear vein. An adjuvant-control rabbit

received the same regimen without crude antigen. Blood was collected

from all rabbits by marginal ear-vein puncture beginning 9 days after

secondary immunization and then every 3-4 days for approximately 7

weeks. Sera collected from individual rabbits was pooled.

Ininunodiffusion

The pooled rabbit antisera and pooled canine sera from dogs

naturally-infected with D. immiiti were used in immunodiffusion assays

to compare adjacent fractions from preparative isoelectric focusing.

The ininunoglobulin portion of each pool was isolated by an ammonium

sulfate precipitation technique (Harboe and Ingild, 1973). Twenty-

five g of ammonium sulfate was added to 100 ml of each serum pool and



Calbiochem, La Jolla, CA

2Bolab, Inc., Derry, Nil

3WyethR, Wyeth Laboratories,Inc., Marietta, PA








this mixture was incubated for 20 hours at 22C. After centrifugation

at 12,100 X g for 30 minutes the supernatant was discarded and the

precipitate was washed twice in 1.75M ammonium sulfate. The washed

precipitate was solubilized with distilled water and dialyzed for 24

hours against distilled water and 24 hours against PBS (pH 7.2).

Adjacent fractions collected from isoelectric focusing were compared

for antigenic activity and identity against the immunoglobulin prepa-

rations of homologous rabbit antisera and canine immune sera using a

standard immunodiffusion technique (Anonymous, 1968). Agarose,1 sodium

chloride and distilled water were mixed in the proportions 1:1:98,and

heated to 90C in a boiling water bath. Twelve ml of the hot solution

was poured onto a level glass plate (9 X 9 cm), then after 2 hours at

4C holes were cut2 in the solidified gel and 10 11 of each fraction

described above was diffused against 10 pJ of purified immunoglobulin

for 72 hours. Nonprecipitating protein was washed from the agarose

with 1% sodium chloride for 48 hours, then the plates were rinsed in

distilled water for 6 hours and dried. Staining was with 0.1% napthyl

blue-black3 in methanol, glacial acetic acid,and water (45:10:45);

destaining was in the same solvent. Adjacent fractions showing antigenic

identity were combined. TSM and TSF were diffused against homologous

rabbit antibody and canine antibody to determine antigenic activity and

complexity.



Biorad Laboratories, Richmond, CA

Gelman Instrument Co., Ann Arbor, MI

Sigma Chemical Co., St. Louis, MO









Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate

Fractions combined after immunodiffusion were subjected to poly-

acrylamide gel electrophoresis in the presence of sodium dodecyl

sulfate (PAGE-SDS) to determine the complexity of each fraction and

the approximate molecular weight of the fraction constituents. The

principle methodology observed was reported by Weber and Osborn (1969)

with adaptations for slab PAGE-SDS (Easterday et at, 1976). Fractions

combined after immunodiffusion were dialyzed for 72 hours at 4C

against 0.001M tris (pH 8) and lyophilized. Five hundred v3 dry weight

of each fraction and 50 ,g of each of 6 protein standards were used.

The standards included cytochrome C, chymotrypsinogen A, ovalbumin,

bovine serum albumin, aldolase,and catalase. Gels were cast in a

gel-casting tower2 in glass cassettes3 (8.2 X 0.27 X 8.2 cm) using

either 7.5%, 10%,or 12% polyacrylamide. The acrylamide solution was

mixed as described (Easterday et al., 1976), poured into the casting

tower and allowed to polymerize for 30 minutes. After polymerization

the gel cassettes were stored in a humidity chamber at 4C for 14-16

hours. Before electrophoresis all samples were treated with 20 pl of

0.01M tris with 1% SDS and 1% 2-mercaptoethanol (2-ME) and incubated

for 15 minutes at 600C. After incubation, 5 pl of a bromophenol blue-

sucrose solution4 was mixed with each sample; 10 samples were applied



1Mol-RangerTM, Pierce Chemical Co., Rockford, IL

Gel Slab Casting Apparatus GSC-8, Pharmacia Fine Chemicals, Piscataway,
NJ

3Gel Cassette Kit, Pharmacia Fine Chemicals, Piscataway, NJ
4 io-phoreTM iorad Laboratories, Richmond, CA
Bio-phore Biorad Laboratories, Richmond, CA








to each cassette. Electrophoresis was performed in a tris-glycine-SDS

buffer at 90V in a specially designed chamber1 until the tracking

dye moved to 1.5-2.0 cm from the bottom of the gel. When electrophoresis

was complete the gels were removed from the glass cassettes, rinsed in

distilled water and fixed in 25% isopropanol, 10% acetic acid for 48

hours. After fixation, the gels were stained for 48 hours in 0.02%

coomassie blue in 7% acetic acid; gels were destined with 25%

isopropanol, 10% acetic acid until background color disappeared. The

distance from the origin to the solvent front was measured as was the

leading edge of each standard band and each distinct band of the

individual fractions. The distance moved (mobility) of each standard

of known molecular weight was plotted to obtain a standard curve for

determining molecular weights of the fraction constituents. A standard

curve for each polyacrylamide percentage was determined. Adjacent

fractions from each of the MSSA, FSSA,and MFSSA with apparent identical

constituents were combined.

Polyacrylamide Gel Electrophoresis

Fractions combined after PAGE-SDS were further examined using PAGE

electrophoresis in 10% polyacrylamide with no SDS. Gels were cast as

described above. Five hundred ug of each sample was solubilized in a

tris-boric acid-EDTA electrophoresis buffer and 6 samples were applied

to each cassette. Electrophoresis was performed at 120V until the

solvent front was 1.5-2.0 cm from the bottom of the gel, then the gels

were removed and split into halves with a wire cutter and gel-slicing



Electrophoresis Apparatus GE-4, Pharmacia Fine Chemicals, Piscataway, NJ








frame.1 Half of each gel was fixed in 20% sulfosalicylic acid for

40 minutes then stained for protein constituents with coomassie blue

as before. The other half of each gel was used to stain for presence

of carbohydrates. Initially, a periodic acid Schiff's stain (PAS)

was used (Maurer, 1971). These gels were fixed in a sodium periodate,

glacial acetic acid, hydrochloric acid and TCA solution for 16 hours,

then washed for 8 hours in a glacial acetic acid, TCA solution and

stained in Schiff's reagent2 for 16 hours. When the PAS stain was

complete, the same gel halves were stained with alcian blue3 in a

0.5% solution in 3% acetic acid for 48 hours; destaining was with 3%

acetic acid.

A general scheme for antigen purification and characterization

is illustrated in Figure 1.

Humoral Immune Response Determinations

Purification of the Indirect Hemagglutination Antigen

Anti-D. inm itis antibodies were measured in all sera collected

using a semipurified antigen prepared according to Sawada et al., (1965)

and Mantovani and Kagan (1967). Adult D. immitis from naturally- and

experimentally-infected dogs were separated by sex, washed 3 times in

PBS,and extracted in 0.01M tris (pH 8) as described above. Equal

protein quantities of each MSSA and FSSA were combined and 10% TCA

was added until the pH reached 3.5. This mixture was left at 4C



Phannacia Fine Chemicals, Piscataway, NJ

Fisher Scientific Co., Fair Lawn, NJ

Canalco, Inc., Rockville, MD
































Figure I. Diagrammatic representation of the scheme used in
purification and characterization of Dirofilaria inrnitis
antigens.









WHOLE WORM


Triton X-100 Solubilized Extract Aqueous Soluble Somatic Extract

/ \\


PAGE-SDS PAGE


Immunodiffusion
Against Sera from
Dir ofi aia izritis-
Infected Dogs


Immunodiffusion
Against Homologous
Rabbit Antisera


Preparative Isoelectric Focusing
into Multiple Fractions





Immunodiffusion Against Homologous
Rabbit Antisera (combine adjacent,
identical fractions)



PAGE-SDS (combine adjacent,
identical fractions)




PAGE








for 1 hour, then centrifuged at 12,100 X g for 45 minutes at 40C;

the supernatant was dialyzed against distilled water for 48 hours at

4C. Approximately 120 mg of TCA soluble protein was lyophilized and

reconstituted to 20 ml with distilled water. Group separation on

this preparation was at 4C, in a glass chromatography column (2.5 X

60 cm) using Sephadex G-1002 equilibrated with distilled water.

Elution was with distilled water at a flow rate of 3 ml/hour. The

eluate was continuously monitored3 for protein at 254 nm and each

5 ml was collected using an automated fraction collector. Those

fractions within the initial, principal protein peak were combined,

lyophilized. and resolubilized with 5 ml of 0.005M sodium acetate

(pH 4.6).

The sodium acetate solubilized preparation was layered onto a

carboxyiethyl cellulose5 column equilibrated with sodium acetate

(pH 4.6) and eluted at a flow rate of 20 ml/hour. The eluate was

monitored and collected as above and after each protein fraction was

completely eluted, the column was eluted with a buffer of increasing

molarity or pH. The eluants used in succession were 0.005M sodium

acetate (pH 4.6), 0.05M phosphate buffer (pH 6), 0.1M sodium chloride

(pH 7), 0.2M sodium chloride (pH 7), D.4M sodium chloride (pH 7), 0.1N



ISCO Chromatographic Column, ISCO, Lincoln, NB

2Sephadex G-100, Pharmacia Fine Chemicals, Piscataway, NJ

UA-5 Absorbance Monitor, ISCO, Lincoln, NB

4Model 328 Fraction Collector, ISCO, Lincoln, NB

5CM-cellulose, Pharmacia Fine Chemicals, Piscataway, NJ








sodium hydroxide (pH 7). The fractions eluted with each of these

eluants were collected separately; previous to use in the indirect

hemagglutination (IHA) assay the fractions eluted with 0.2M and 0.4M

sodium chloride were combined and dialyzed against PBS (pH 7.2) for

48 hours at 4C. The protein concentration of this preparation was

determined previous to antigen titration. A scheme for the purification

of the IHA antigen is illustrated in Figure 2.

Indirect Hemagglutination Assay

The IHA method was a modification of a widely used technique in

the serodiagnosis of parasitic diseases (Kagan and Norman, 1976).

Approximately 5 ml of sheep red blood cells (SRBC) suspended in Alsever's

solutionI were washed 3 times in hemagglutination buffer (HAB)2 (pH 7.3).

Centrifugation was for 10 minutes each time at 800 X g.3 After the

final wash, the packed cells were adjusted to a 2.5% suspension in HAB

and mixed with an equal volume of 1:2 X 104 tannic acid solution. This

mixture was incubated at 37C for 10 minutes, then the cells were washed

twice and resuspended to a 2.5% solution with HAB. The tanned cells

were sensitized with one of five dilutions (1:4, 1:8, 1:16, 1:32, 1:64)

of the column purified antigen by adding an equal volume of the antigen

dilution to a 2.5% suspension of tanned SRBC; the mixture was incubated

at 37C for 15 minutes. The antigen-sensitized SRBC were washed twice

and adjusted to a 1.5% suspension with HAB with 1% heat inactivated



Becton, Dickinson and Co., Cockeysville, MD

2Bacto Hemagglutination Buffer, Difco Laboratories, Detroit, MI

3PR-2 Refrigerated Centrifuge, International Centrifuge Co., Needham
Heights, MA











Adult D.irof'iaria imnit-is


Aqueous Soluble
Somatic Extract



Trichloroacetic Acid
Treatment


Discard Precipitate Supernatant Dialyzed
vs. Distilled Water



Gel Filtration-GlO0



Cation-Exchange Chromatography
on Carboxyimethyl Cellulose

.005M .05M .1 .4 M .1 M
Acetate Buffer Phosphate Buffer NaC1 NaCI NaC1 NaOH
pH 4.6 pH 6 pH 7 pH 7 pH 7 pll 7


Indirect Hemagglutination
Antigen





Figure 2. Diagrammatic representation of the
procedure for purification of the
Dirofi laria imunitis indirect
hemagglutination antigen








fetal calf serum (FCS). Twenty-five iIl of the diluent, HAB with

1% FCS, was added to each well of a V-bottom microtiter plate2 in

which serum dilutions were to be made. Fifty pl of either pooled

canine immune serum or pooled canine serum from 4 dogs not infected

(normal serum) were serially diluted with 25 pl dilution loops.

When the serum dilutions were complete, 25 il of each antigen-

sensitized SRBC suspension was added to each well in a dilution series

of both normal and immune serum. The plates were incubated for 3

hours at 220C; the highest serum dilution with hemagglutination was

considered the titer. The antigen dilution giving the highest titer

with the immune serum and no reaction with the normal serum was

optimal; this dilution was used to determine anti-D. imnitis antibody

titers in individual serum samples collected over the course of the

investigation. A diluent control was made by adding 25 1l of diluent

and 25 p1 of antigen-sensitized SRBC to 12 individual wells; a tanned

SRBC control was made by adding 25 pl of unsensitized, tanned SRBC

to each well of a dilution series of both normal and immune serum. In

both controls it was necessary to obtain negative reactions. Differ-

ences in anti-D. imranibis antibody titers of single infected, double

infected,and noninfected dogs were statistically evaluated using a

general linear models procedure (Steel and Torre, 1960).



1Grand Island Biological Co., Grand Island, NY
Cooke Laboratory Products, Alexandria, VA

Beckman Instruments, Inc., Palo Alto, CA








Cell-Mediated Immune Response Determinations

Lymphocyte Isolation

The lymphocyte isolation technique was a modification of the method

of Thilsted and Shifrine (1977). Fifteen ml of blood was obtained by

venipuncture from the cephalic vein of each dog and mixed with heparin

(10 U/m1 of blood). The anticoagulant treated blood was mixed with

15 ml of RPMI-1640; this mixture was divided in half, layered over 30 ml
2
of a sucrose polymer-diatrizoate solution with 0.1% methylcellulose.

The tubes were centrifuged at 800 X g for 30 minutes at 220C. The

resultant lymphocyte-rich layer was harvested with a sterile pipette

and washed in 50 ml of RPMI-1640. Total leuckocyte counts were made

using a hemacytometer, then 200 pl of the preparation was fixed on a
3 4
microscope slide with a cyto-centrifuge, stained, and examined

microscopically to obtain a differential leuckocyte count. Leukocytes

were classified as either mononuclear or polymorphonuclear. After

total mononuclear cell numbers were determined each cell preparation

was washed in 50 ml of RPMI-1640 and resuspended to 3.0 X 106 mono-

nuclear cells/ml in RPMI-1640 supplemented with 1% antibiotic-

antimycotic5 and 10% normal canine serum absorbed with human red blood

cells (HRBC). This cell suspension was used for the lymphocyte-rosette

assay, then adjusted to 1.25 X 106 mononuclear cells/il for lymphocyte

transformation.



Grand Island Biological Co., Grand Island, NY

2LSMTM Solution, Bionetics Laboratory Products, Kensington, MD

Shandon-Elliott, Sewickley, PA

Camco Quick Stain, Scientific Products, McGraw Park, IL

Grand Island Biological Co., Grand Island, NY








Lymphocyte Transformation

Four major lymphocyte transformation experiments were performed

43, 45, 49, and 50 weeks after the initial D. irniitis infection. All

experiments were initiated after patency of the first infection and

after Group C was infected the second time.

Two-tenths ml of each cell suspension was placed into each

predetermined well in a microculture dish1 to give 2.5 X 105 cells/well.

Various levels of mitogen and antigen were assayed for lymphocyte

transforming ability in quadruplicate cultures; unstimulated cultures

from each cell preparation served as a background control. Phyto-

hemagglutinin P2 (PHA) was added in a range of 0.025-0.2 pl/culture,

and Pokeweed Mitogen3 (PWM) and Concanavalin A4 (Con A) were added in

ranges of 1-10 pl/culture and 1-10 pg/culture, respectively. The

D. imnitis antigen used was a combination of MSSA and FSSA (1:1) in

PBS (pH 7.2) and was used in a range of 10-100 jg of protein/culture.

All mitogens and antigens were solubilized in RPMI-1640 and were added

to the respective cultures in a 10 vl volume. The cultures were

incubated5 for 48 hours at 370C in 5% CO2, then 0.5 ,Ci of tritiated

thymidine in RPMI-1640 was added to each well and incubation was

continued under the same conditions for 16-20 hours. When the incubation



Cooke Laboratory Products, Alexandria, VA

Difco Laboratories, Detroit, MI
3
Grand Island Biological Co., Grand Island, NY

Miles Laboratories, Inc., Elkhardt, IN

5Model C0-20, New Brunswick Scientific, New Brunswick, NJ

6The Radiochemical Centre, Amersham, England







was complete, the cultures were collected individually on a paper strip

using an automated culture harvester. The strip was air-dried and each

portion of the paper with the cells from an individual culture was

placed in a scintillation vial. The vials were filled with 4.5 ml of
2
a scintillation cocktail; after 3-6 hours of dark adaptation the

radioactivity in each vial was measured using an automated liquid

scintillation counter.3 The amount of tritiated thymidine incorporated

in cultures at each level of mitogen or antigen treatment was expressed

as the mean counts per minute (cpm) of the 4 replications at each level.

A stimulation index (mean cpm of stimulated culture/mean cpm of non-

stimulated culture) was used to evaluate the extent of lymphocyte

transformation.

Preliminary evaluation of lymphocyte transformation data indicated

that there was no difference between dogs receiving single or double

D. inrnitis infections; all data on cell-mediated immune response

determinations are expressed as noninfected dogs (Group A) and infected

dogs (Groups B + C). Further, because of limited animal numbers and

the variability in lymphocyte responses it was necessary to evaluate

the data after all 4 lymphocyte transformation experiments were combined.

Additionally, data on the lymphocyte responses to all levels of each

antigen and mitogen were combined to evaluate differences in lymphocyte

responsiveness. Differences in mitogen and antigen responses were

analyzed using a general linear models procedure.



Otto Hiller Co., Madison, WI

2Fisher Scientific, Pittsburg, PA

LS 330, Beckman Instruments, Inc., Palo Alto, CA








Lymphocyte Rosette Assay

A modification of the procedure of Bowles et at. (1975) was used

to quantitate canine lymphocytes forming nonimmune erythrocyte rosettes

with HRBC. This assay was performed with the same cell preparations

used in the 4 lymphocyte transformation experiments. Two-tenths ml

of a 0.5% suspension of HRBC in RPMI-1640 and a 0.2 ml aliquot of each

lymphocyte suspension at 3.0 X 106 cells/ml were mixed in triplicate.

This mixture was incubated at 22C for 30 minutes then centrifuged at

200 X g for 5 minutes. The cell pellet was incubated at 4C for 14

hours then gently resuspended; one drop of the suspension was mixed

with one drop of a dilute acridine orange stain (Brostoff, 1974). The

stained suspension was examined in a hemacytometer at 100X using

fluorescent microscopy. One hundred fluorescing cells were counted

and cells binding two or more erythrocytes were counted as rosette-

forming cells. The percentage of rosette-forming cells in each cell

suspension was expressed as the mean of the triplicate samples.

Immune Response to Sheep Red Blood Cells

After the initial data on the lymphocyte transformation experiments

were obtained, it was apparent that a comparison of the immune response

of infected and noninfected animals to a heterologous antigen, SRBC,

would be advantageous. Three ml of a 20% SRBC suspension in PBS (pH 7.2)

was administered intravenously to all dogs 49 weeks after the initial

D. uinniti infection and again 16 days later. Sera was collected on

the day of the primary immunization and 8, 16, 20, 22, 27, and 29 days

thereafter for anti-SRBC antibody determinations. Antibody was measured


Eastman Kodak Co., Rochester, NY








by direct hemagglutination. Two 25 il aliquots of each serum sample

were mixed with either 25 jil of HAB with 1% FCS or 25 1l of 0.1M 2-ME

and incubated at 37C for 1 hour (Scott and Gershon, 1970). After

incubation, the sera was diluted in a microtiter system as described

above and 25 pl of a 1.5% solution of SRBC was added to each well.

Titers for untreated and 2-ME treated sera were determined after

incubation at 220C for 3 hours. This assay was performed twice on

each serum sample and the mean titer was recorded.

One hundred il of sera from each dog 20 and 22 days after primary

SRBC immunization was pooled and approximately 2 ml of this pool was

separated by gel filtration on a Sepharacyl S-2001 column (2.5 X

80 cm). The Sepharacyl S-200 was equilibrated and eluted with a

sodium chloride, trizma base, hydrochloric acid buffer. The column

flow rate was approximately 5 ml/hour and each 2.25 ml was collected

in individual tubes on an automatic fraction collector.2 Ultraviolet

light absorbance at 280 nm of the eluate in each tube was determined

and 4 pools corresponding to the manufacturer's predicted peaks for

IgG, IgA, IgM and albumin fractions were collected. Each pool was

concentrated with negative-pressure dialysis then assayed for 2-ME

labile antibody as described above to insure that IgM was the only

fraction demonstrating 2-ME ability. Differences in anti-SRBC antibody

and 2-ME labile anti-SRBC antibody between infected and noninfected

dogs were evaluated using a general linear models procedure.



Pharmacia Fine Chemicals, Piscataway, NJ

Gilson Medical Electronics, Inc., Middleton, WI














RESULTS

Fecal examinations for helminth ova on all dogs were negative

before and throughout the experimental period. All dogs remained

healthy except for occasional minor lacerations obtained during

fighting.

All dogs in Groups B and C received 29-30 infective larvae each.

Dogs in Group C were infected with an additional 30 larvae each at the

second D. innitis infection.

Microfilariae Counts

Microfilariae were first detected 26 weeks after infection in one

dog and were present in all infected dogs by 30 weeks after infection

(Appendix I). One dog that received 2 D. irrnitis infections became

amicrofilaremic 56 weeks after the initial infection and 22 weeks after

the second infection; microfilariae were not detected in that dog again

throughout the study (Appendix I). A marker dog was not used to detect

the onset of patency of the second infection; there was no evidence of

a difference in microfilariae counts between single and double infected

dogs at any time (Figure 3).

Antigen Purification and Characterization

Preparative Isoelectric Focusing

The preparative, flatbed isoelectric focusing effected a good

protein separation with a reproducible pH gradient and approximately

a 70M protein recovery of the crude soluble somatic preparations.

MSSA, FSSA,and MFSSA were initially divided into 17, 22,and 9 fractions,




























Figure 3. Mean numbers (X103) of Dirofilaria imrrmtis micro-
filariae per ml of blood in Group B (single
infection) and Group C (double infection).


















































/ ~-~-


(O x C C R I- sDil.'- N ON NjAA


ID








-w


ID











U
C I








respectively, based on the isoelectric points and the staining intensity

of apparent protein bands on the filter paper print. The filter paper

print of each separation, the percentage of total recovered protein

in each final fraction,and the pH gradient established during separation

are illustrated in Figures 4-6.

Immunodiffusion

After immunodiffusion of the fractions separated by isoelectric

focusing, the male, female,and microfilaria fractions were combined

to 11, 14,and 6 fractions, respectively. If antigenic identity could

be established, adjacent fractions were combined. Antigenic activity

against both homologous rabbit antibody and canine antibody was

present in most fractions (Tables 1-3). Adult worm fractions with the

most apparent antigenic activity and complexity were within an iso-

electric point (pl) range of approximately 4.8-6.5.

Approximately 4 mg of TSF and 2 mg of TSM were recovered. There

was some apparent antigenic activity when these were diffused against

homologous rabbit antibody and canine antibody, but precipitin lines

were weak and the degree of antigenic complexity could not be discerned.

Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate

Adjacent fractions of each preparation with apparently identical

protein constituents after PAGE-SDS were combined after consideration

of isoelectric point ranges and antigenic identity. The male, female,

and microfilaria fractions were combined to 7, 7, and 3 fractions.

respectively, the final fraction number for each preparation. The

approximate molecular weights of the protein constituents in the soluble

somatic fractions are listed in Tables 1-3. The TSM and TSF prepa-

rations were less complex. The TSM had 2 protein constituents of































Figure 4. Preparative isoelectric focusing separation and
percentage of recovered protein in the final
fractions from the Divoilaria immitis male
soluble somatic extract.























(o io


N13IONd 0383A038 O 39VIN3063d


oiSWI~* ~'.































Figure 5. Preparative isoelectric focusing separation and
percentage of recovered protein in the final
fractions from the Dirofilaria irnitis female
soluble somatic extract.











































































Nl3108d (O383A03D8 AO 3VlN1081d


9

~-a

4































Figure 6. Preparative isoelectric focusing separation
and percentage of recovered protein in the
final fractions from the Dirofilaria irmnitis
microfilaria soluble somatic extract.























PERCENTAGE OF RECOVERED PROTEIN


p-.











TABLE 1. Diroj'iuaria imr-icis male soluble somatic antigen fractions purified by
preparative isoelectric focusing and characterized by immunodiffusion,
polyacrylamide gel electrophoresis,and polyacrylamide gel
electrophoresis in sodium dodecyl sulfate.


Constituent
Fraction pi Range Proteins after
PAGE


Constituent
Proteins after
PAGE-SDS


Mol. Wt. of
Constituent4
Proteins (X10)


Antigenic Activitya
Ra. anti- Ca. anti-
MSSA D. initis


M-1 3.65-4.35


M-2 4.60-4.83


M-3 4.93-5.35


M-4 5.43-6.10



M-5 6.20-6.40


M-6 6.57-7.80


M-7 8.23-8.50


77


>100,56,18


7 90,68,56,52,43,30,18


9 68,56,52,43,36,30,26
24,17.5


6 68,43,30,26,20,17.5


68,30,26,20


7 >100,>100,>100,74
43,20,17.5


aAntigenic activity was determined by


immunodiffusion.











TABLE 2. Dirofi aiia irrnitis female soluble somatic antigen fractions purified by
preparative isoelectric focusing and characterized by immunodiffusion,
polyacrylamide gel electrophoresis,and polyacrylamide gel
electrophoresis in sodium dodecyl sulfate.


Constituent
Fraction pl Range Proteins after
PAGE


Constituent
Proteins after
PAGE-SDS


Mol. Wt. of
Constituent3
Proteins (X10)l


Antigenic Activitya
Ra. anti- Ca. anti-
FSSA D. -initis


F-l 3.75-4.40


F-2 4.50-4.85



F-3 5.00-5.73



F-4 5.93



F-5 6.15-6.45



F-6 6.49-7.70


F-7 8.00-8.49


10 >100,82,77,59,42,36,33,
31,19,18


11 >100,85,77,68,59,42,
36,33,24,21,18


10 85,68,59,42,36,33
30,24,21,18


8 >100,77,68,42,33,24,
21,19


8 90,77,68,52,35,29,25,21


8 >100,>100,90,77,52
35,25,21


aAntigenic activity was determined by immunodiffusion.












TABLE 3. Dirofilaria mminris microfilaria soluble somatic antigen fractions
purified by preparative isoelectric focusing and characterized by
irnunodiffusion, polyacrylamide gel electrophoresis,
and polyacrylamide gel electrophoresis in sodium dodecyl sulfate.


Constituent
Fractions pi Range Proteins after
PAGE


Constituent
Proteins after
PAGE-SDS


Mol. Wt. of
Constituent
Proteins (X10)l


Antigenic Activitya
Ra. anti- Ca. anti-
MFSSA D. i nitis


MF-l 3.55-4.20 4 1


MF-2 4.30-6.25 2


MF-3 6.45-9.35 1 1



aAntigenic activity was determined by immunodiffusion








molecular weight 7.7 and 5.2 X 10 ; the TSF had 5 protein constituents of

molecular weight 7.7, 7.1, 6.6, 5.2,and 3.4 X 104. Photographs of the

separated fractions on polyacrylamide gel slabs appear in Appendix II.

The standard lines for molecular weight determination obtained for

each polyacrylamide gel percentage are in Appendices III-V.

Polyacrylamide Gel Electrophoresis

The number of proteins detected in individual fractions after

PAGE are listed in Table 1-3. Two protein bands from the TSM prepa-

ration and 5 from TSF preparation were detected. Neither the PAS

stain or the alcian blue stain were successful in detecting carbo-

hydrate moieties associated with any of the separated protein

constituents. The sample application point of all fraction and the

electrophoresis tracks of the crude male and female preparations

appeared to react with both stains, but staining was diffuse and could

not be related to constituents of any of the fractions. Photographs

of the PAGE separated soluble somatic fractions, crude soluble somatic

preparations and Triton X-100 solubilized preparations are in Appendix VI.

Humoral Immune Response

Thirty mg of the column-purified 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. A

titer of 1:512 with pooled canine immune sera and no titer with pooled

canine normal sera were repeatedly obtained with SRBC sensitized with

this antigen dilution.

Anti-D. immnitis antibody was first detected in Groups B and C

4 weeks after the initial D. inmiitis infection (Figure 7). Antibody






























Figure 7. Mean log2 of the reciprocal anti-Dirofilaria innritis
antibody titers in Groups A, B and C over the
course of infection.














- 4,- -- = GROUP A (U.JINFECTED)
F-
= GROUP B (SINGLE INFECTION)
-- --3GROUP C (DOUBLE INFECTION)
5 3L- 1


E 3.0- U



iI\C
S/ 1\


SA\ / "\ i \
S ,i v/\' /'t //

,o 'i u/ \ 1 / \-'\ / / \ .'..

S\ \ CON IINFECTIO
GROUP C)A A A


-4 0 4 8 12 16 20 24 28 32 36 40 44
WEEKS AFTER INITIAL D mmi ts INFECTION


52 56 60 64









levels in the infected groups were significantly higher (p<0.05) than

in the uninfected group. The antibody titer in Group B, the single

infection group, began to decrease shortly after patency (Week 30)

and antibody levels in Group C, 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. Individual anti-D. immitis

antibody titers are tabulated in Appendix VII.

Cell-Mediated Immune Response

Lymphocyte TransFormation

Peripheral blood lymphocyte transformation could not be induced

in D. in,?i tis-infected dogs with any level of D. imnitis antigen tested

(Table 4). A significantly (p<0.0001) depressed reactivity to PHA,

PWM,and Con A was observed in lymphocyte cultures from infected dogs

(Table 4).

Lymphocyte Rosettes

There were no differences evident in the mean percentage of

rosette-fonring cells between infected and noninfected dogs (Table 5).

Although the standard deviation observed was low, and the mean percentage

of rosettes remained reasonably consistent between experiments, the

percentage of rosette-forming cells from the same dog often differed

markedly between experiments.

Imiwune Response to Sheep Red Blood Cells

There were no significant differences in anti-SRBC antibody levels

between infected and noninfected dogs (Figure 8). Although there

appeared to be a greater quantity of anti-SRBC 2-ME labile antibody

after the secondary infection (Figure 9), it was not statistically







TABLE 4. Mean counts per minute and stimulation
indices from four canine peripheral blood
lymphocyte transformation experiments of Dirofilaria immitis-
infected dogs and noninfected dogs.

Culture Group A Groups B + C
Treatment (noninfected dogs) (infected dogs)


none 84a 90

.025pl PHA 2152 (25.6)b 1263 (14.0)

.05y1 PHA 4676 (55.7) 2588 (28.8)
*
.0lp PHA 7103 (84.6) 3740 (41.6)

.20ul PHAc 8607 (102.5) 4575 (50.8)

1pl PWM 3650 (43.5) 2129 (23.7)

541 PWM 4164 (49.6) 2367 (26.3)

10p PWM 3731 (44.4) 2132 (23.7)

lug Con Ad 5961 (70.9) 2741 (30.5)

5pg Con A 8501 (101.2) 5090 (56.6)

10g Con A 9454 (112.6) 4329 (48.1)

10l g DIAe 96 (1.2) 90 (1.0)

50pg DIA 118 (1.4) 104 (1.2)

100pg DIA 103 (1.2) 100 (1.1)

*
Statistically significant difference (p<0.0001)
aMean counts per minute (cpm)

bStimulation index (cpm of stimulated culture/cpm of nonstimulated
culture
This level was evaluated in one experiment

This mitogen was evaluated in half of the dogs in each group in
one experiment
eDirofla2i imm-i tis antigen







TABLE 5. Percentage of canine peripheral lymphocytes
forming nonimmune rosettes with human erythrocytes.


Experiment


Group A
(noninfected dogs)


24.7 + 5.5a

18.2 + 1.8

26.7 + 5.2

19.9 + 5.5


Groups B + C
(infected dogs)


24.6 + 5.7

33.2 + 5.8

31.6 + 4.8

28.8 + 5.8


Data is expressed as the mean leukocyte percentage of rosette-
forming cells + the standard deviation of triplicate samples
from each dog.





























Figure 8. Mean log, of the anti-sheep red blood cell
antibody titers in Group A and Groups B + C.















.35 // 7

o/ \
30



S25- \
-/ \V

SECONDARY SR.B.C IMMUNIZATION
S20- /
o00
/ -- = GROUP A (UNINFECTED)
-- = GROUPS B+ C(INFECTED)
o /
.0-

1.0
S- /


DAYS AFTER PRIMARY S.R.B.C IMMUNIZATION































Figure 9. Mean difference of log2 of reciprocal anti-sheep
red blood cell antibody titers before and after
serum treatment with 2-mercaptoethanol.















[-


S/ GROUP A (UNINFECTED)

S\ I GROUPS B-C INFECTED)



0s 35 /


_J / 1





S25-


o SECONDARY SR C
o 2 IMMUNIZATION




S 15-
ll '
<


5 10 15 20 25

DAYS AFTER PRIMARY SR B C IMMUNIZATION








significant. The average anti-SRBC antibody titers before and after

2-ME treatment for each dog are listed in Appendix VII. A good

separation of the pooled anti-SRBC on Sepharacyl S-200 was effected

(Figure 10). Anti-SRBC antibody activity was detected in Pools A,

B,and C. Pool A and Pool C demonstrated the predominance of anti-

SRBC activity. After 2-ME treatment the mean anti-SRBC antibody

titer decreased from 1:8 to 1:1.5 in Pool A, the IgM pool. Antibody

titers in Pool B, the IgA pool, and Pool C, the IgG pool, remained

unchanged after 2-ME treatment.






























Figure 10. Separation profile of pooled canine anti-sheep
red blood cell serum on Sepharacyl S-200 and
the pools collected for 2-mercaptoethanol
ability determinations.














POOL 0
POOL
-


SEPHACRYL S-200
POOLED CANINE ANTI SR B C.


TUBE NUMBER















DISCUSSION

Antigen Purification and Characterization

Any definitive conclusions on the antigenic relationships of

different D. immtitis life-stages are not possible when the data from

this investigation are evaluated; however, this study contributed to

progress toward that end. The degree of antigenic complexity detected

in the different life-stages is remarkable and with the partial-

characterization data it will be helpful when further studies on

antigenic relationships are considered.

Separating the myriad of proteins present in each crude soluble

somatic extract with preparative isoelectric focusing proved to be

advantageous. Recent reports have described disc electrophoresis,

immunoelectrophoresis and countercurrent immunoelectrophoresis

techniques that were successful in delineating the protein,and antigenic

complexities of crude D. immitis extracts (Wheeling and Hutchison,

1971; Desowitz and Una, 1976). The principal deficit in the findings

in both reports was that the separated and partially characterized

fractions could not be directly related to immunological activity.

In the present study, the fractions recovered after isoelectric

focusing of large quantities of protein were easily harvested and

could be used individually in iimiunological and biochemical characteri-

zation techniques.

There were antigens in all of the adult fractions and 2 of the

3 microfilaria fractions. The antigens were demonstrable using either









homologous rabbit antisera or canine immune sera; the predominance

of antigenic activity and complexity in the adult worm fractions was

in the pi range of approximately 4.8-6.5. In any further studies,

the ampholines used in preparative isoelectric focusing could be

confined to this pH range to concentrate on a more complete separation

in this area. Since immunoelectrophoresis demonstrated at least

10 precipitin arcs in the crude adult D. imnitis preparation tested

against homologous rabbit antisera (Wheeling and Hutchison, 1971),

further separation of the soluble somatic fractions will be necessary

before antigenically distinct fractions are obtained.

PAGE-SDS was effective in determining approximate molecular

weights as well as comparing the protein-staining constituents of

adjacent fractions. Although there was some contamination evident in

the proteins used in establishing the standard lines for molecular

weight determinations, reliable lines could be established. The lowest

molecular weight standard, cytochrome C, traveled into the solvent

front in the 7.5% polyacrylamide resulting in a point inconsistent

with the standard line (Appendix III). The 2 lowest molecular weight

standards did not fit the standard line well in the 12% gels (Appendix

V), but the line was consistent in the molecular weight range of most

proteins present in the fractions. It is likely that the 12% poly-

acrylamide had too much cross-linking bis-acrylamide resulting in this

effect (Weber and Osborn, 1969).

Stains for carbohydrate after PAGE were unsuccessful. Some diffuse

staining with PAS and alcian blue was noted in the crude fractions;

the staining appeared to be dispersed over the entire electrophoresis track







in each case. Additionally, there was some staining evident at the

application point of each fraction. Wheeling and Hutchison (1971)

reported 2 bands of carbohydrate-staining activity after disc electro-

phoresis of a crude adult extract; one band was near the origin and one

was near the solvent front. It is possible that disc electrophoresis

yielded sharper, more dense bands than the PAGE resulting in a more

confined, intense stain.

It was unusual to note that in fraction F-1 and MF-1 more protein

constituents were detected after PAGE than after PAGE-SDS. The 2-ME

reduction before PAGE-SDS may have reduced large macromolecules to a

number of small molecular weight proteins that traveled with the solvent

front or different macromolecules may have had constituents of similar

molecular weight that moved a common distance after reduction and PAGE-SDS.

The Triton X-100 solubilization of adult worm cuticles was

largely unsuccessful. Unfortunately, only limited quantities of

protein could be recovered. These preparations contained few proteins;

relatively only a few bands appeared on PAGE and PAGE-SDS gels. Both

preparations appeared to be weakly antigenic against both homologous

rabbit antisera and canine immune sera when compared to soluble somatic

preparations. Some success with Triton X-100 solubilization has been

reported with Onchocerca volvulus antigens (Marcoullis and Grasbeck,

1976), but it was not clear if the worm preparations were thoroughly

extracted in an aqueous system before Triton X-100 solubilization. It

is possible that some of the antigenic complexity reported was due to

residual aqueous soluble proteins.

Future work on the characterization of these soluble somatic

antigen fractions should make use of immunologic techniques. After








having gained some information on the protein makeup, comparisons of

the antigenic components of the fractions are necessary to learn

about the possible stage- or sex-specific antigens. Antigenic

comparisons of each fraction with every other fraction could be

considered, but initially it would be important to compare those

fractions from different preparations within the same isoelectric point

range. Comparative immunodiffusion would be a logical technique to

use in making the comparisons, but interpretation may be difficult.

A radioimmunoassay technique has been successfully used in the

determination of stage and species specificity of Schistosoma mansoni

antigens (Hamburger et at., 1976). This technique would be ideal

for antigenic comparisons of the D. innitis fractions, but a greater

degree of purity in the fractions will be necessary before the technique

could be implemented.

Humoral Immune Response

Results obtained on antibody titers were largely comparable to

data from other similar studies (Pacheco, 1966; Weiner and Bradley,

1972). Anti-D. imamitis antibody was detectable between 2 and 4 weeks

after infection and peak titers were reached 2 weeks after D. inmitic

reached patency in all dogs. It would be difficult to interpret, as

Pacheco (1966) did, that microfilariae may be responsible for

absorbing antibody and removing it from peripheral circulation, but the

onset of the microfilaremia is obviously associated with the decrease

in circulating antibody. Since the titers diminish 2-4 weeks after the

appearance of microfilariae, the microfilariae may be directly affecting

the immune system and the decline of antibody titers could be due to









the normal, biological half-life of the specific immunoglobulins.

Weiner and Bradley (1972) reported that a second D. iniitis infection

did not result in a typical anamnestic response, although dogs infected

a second time had prolonged antibody levels when compared to dogs with

single infections.

The results illustrated in Figure 7 indicated that there was a

delayed anamnestic response in the group receiving 2 infections, but

that response was not statistically significant (p>0.05). On close

observation, this apparent secondary response was related to

unusually high antibody titers in the dog that became amicrofilaremic

(Appendix VII). This animal developed high antibody titers at Week

50, just prior to the amicrofilaremic state and at the same point of

the apparent anamnestic response. The unusual antibody response

in this animal accounts for the artifact (Figure 7) when the antibody

levels are illustrated; it also contributes to the enigma of the

relationship of the microfilariae to the canine immune response during

the progress of infection.

Mantovani and Kagan (1967) reported that the TCA soluble antigen

used in the present investigation was both genus- anc species-specific

based on IHA and skin testing of naturally-infected dogs and Pacheco

(1966) reported that an acid-soluble preparation of saline extracted

adult D. inibis was highly specific in serologic testing of experimentally-

infected dogs. It is impossible to assess the specificity of the purified,

acid-soluble antigen preparation used without testing it against sera

from dogs or other animals infected with other parasites, but evidence

indicates that it is more specific than the crude preparations typically








used. The IHA titers for noninfected dogs reported by Weiner and

Bradley (1972) were often as high as 1:256 with a crude saline-soluble

antigen,making interpretations of antibody changes very difficult.

On this basis alone, the purified antigen used in the present study

was more specific; anti-D. inonitis antibody titers of the noninfected

animals never exceeded 1:2. Further, the antigen preparation 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 and Bradley, 1972).

Based on these results, future work on the humoral immune

response to D. immitis should be concentrated on a more specific

and well-defined antigen and a better serologic test. As research

progresses on the isolation and characterization of D. immitis antigens

a stage-specific antigen may be obtained. Detection and quantitation

of adult and microfilaria-specific antibodies over the course of

infection may answer questions on the decrease in antibody after the

appearance of microfilaria and on the nature of the antibody response

proceeding the amicrofilaremic state in some dogs. The enzyme-linked

immunoabsorbent assay has been used in serologic testing of Onchocerca

volvulus (Bartlett et al., 1975), another filarid parasite; if it is

as sensitive and reproducible as reported for other systems (Voller

et at., 1976), it may be ideal for antibody determinations in D. imnmtic

infections.

Cell-Mediated Immune Response

No evidence of D. irmitis antigen-induced lymphocyte transformation

of peripheral blood lymphocytes of infected dogs was obtained. In








addition to the antigens used in the 4 major lymphocyte transformation

experiments, the acid-soluble IHA antigen and a crude 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 et at., 1977; Portaro et al., 1977). Ottesen et al.

(1977) used antigens from saline extracted D. inmitis and Brugia malayi

adults to successfully transform peripheral blood lymphocytes from humans

infected with Wuchereria bancrofti. Mean stimulation indices as high

as 36 were recorded when D. immitis antigens were used to transform

peripheral blood lymphocytes of noninfected, W. bancrofti-exposed

patients. Portaro et al. (1977) have used saline extracts of D. immitis,

Trcivhnella spiraZis and Brugia pahanci to transform splenocytes from

B. pahangi infected jirds. Each of the filarial antigen preparations

induced lymphocyte transformation but the T. spiralis antigen did not.

Of the 2 filarial antigens, the B. pahangi preparation was more effective

in transformation. These data suggested a possible filaria-specific

diagnostic test and confirmed that specific antigen-induced lymphocyte

transformation was possible in a filarial system.

The failure to induce D. i,,aitis-specific lymphocyte transformation

in this study may have been related to a highly significant (p<0.0001)

depression of mitogen responsiveness in infected dogs. Depressed

mitogen responses have been reported in such diverse disease situations

as cancers (Mannick eL al., 1977), leishmaniasis (Farah et at., 1976),

and malaria (Spira at al., 1976). Several nonfilarial helminths have

been associated with an immune suppression phenomenon including T. spiralis








(Cypess et al., 1973; Faubert and Tanner, 1975), Nematospiroides dubious

(Shimp ct at., 1975), Ancaris suwn (Crandall and Crandall, 1976),

Tacnia crassiceps (Good and Miller, 1976), and Schistosoma mansoni

(Pelley at at, 1976). Two diseases of dogs, demodectic mange (Scott,

et at, 1974 and 1976) and canine distemper (Krakowka et al., 1975) have

been associated with diminished mitogen responsiveness. Scott et al.

(1974) reported a correlation between low mitogen responsiveness and

U)emodex canis infection and later work (Scott el at., 1976) indicated

that successful treatment for removal of the mite would reverse the

phenomenon. Recently, various types of ininune suppression have been

documented for 3 other filarid parasites (Dalesandro and Klei, 1976;

Ottesen et la., 1977; Portaro et al., 1976). Dalesandro and Klei

(1976) using Diprtalonima vitae 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. Ottesen et at. (1977) claimed that a specific cellular

unresponsiveness to w. bancrofti occurred in infected humans.

Humans with infections, humans without infections but exposed to the

parasite.and humans with no exposure to the parasite were immunized

with tuberculin (PPD) and streptococcal (SK-SD) antigens. Lymphocyte

responses to those antigens, filarial antigens and mitogens revealed

that only the response to filarial antigens was impaired in infected

humans; mitogen, PPDand SK-SD responses were all normal. 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).

It is possible that D. imnniis antigen-induced lymphocyte transfor-

mation may have been observed if such experiments were attempted before

patency; evidence (Dalesandro and Klei, 1976) would indicate that the

immune suppression phenomenon may be associated with the microfilaremia.

The decline in anti-D. imni-tis titers shortly after the onset of the

microfilaremia would support the idea of a microfilaria-associated

immune suppression. Further work should concentrate on the role of the

microfilariae in this effect.

Since the 3 mitogens used in this study were either T-cell mitogens

or T-cell dependent B-cell (thymus-independent lymphocyte) mitogens in

other organisms, an impaired T-cell function or a decreased number of

T-cells in the infected animals would explain the reduced responses.

Enumeration of canine T-cells by nonimmune rosette formation with HRBC

was reported (Bowles et al., 1975) and this technique was implemented

in this investigation. There were no differences in the percentage of

rosette-forming cells between infected and noninfected dogs. Recent work

(Krakowka and Guyot, 1977) demonstrated that canine eosinophiles formed

nonimmune rosettes with HRBC. In the rosette procedure used in this

investigation, acridine orange stain was used for ease of counting the

cells. At a magnification of 100X leukocyte nuclear morphology was not

easily determined thus a small percentage of the cells counted as lymphoid

rosettes may have actually been eosinophilic leukocytes. Experiments

were performed to examine the rosettes at high magnification and the

results confirmed the work of Krakowka and Guyot (1977). The slightly








higher percentage of rosette-forming cells in infected dogs was

probably due to more contaminating eosinophiles in the mononuclear

cell preparation because of the chronic eosinophilia (Weiner and Bradley,

1972) in D. inm'itis-infected dogs.

In an attempt to assay T-cell function in D. imzittis-infected

dogs a heterologous T-cell dependent antigen, SRBC, was used to

immunize the dogs and measure potential differences in antibody responses

to that antigen. There were no differences between the groups in

total hemagglutinating antibody or 2-ME labile hemagglutinating antibody.

The 2-ME labile antibody responses seemed to occur primarily in the

IgM class of immunoglobulin after secondary immunization of infected

dogs and in the IgG class in noninfected dogs. This observation would

support the hypothesis of impaired T-cell function, but it could not

be substantiated with statistical analysis. Unfortunately, it was not

possible to do antigen dose response studies of SRBC in the canine

before immunization and the dose may have been too high; T-cell

dependence or independence of the immune response to this antigen has

been shown to be antigen concentration-dependent (Playfair and Purves,

1971; Lemmel at ac., 1971).

The implications of a state of diminished immune responsiveness

in D. inm,,tis-infected dogs are numerous. This phenomenon must be

considered in evaluating the pathogenesis of D. ilmmiti, infections. It

is reasonable to assume that this condition will predispose an infected

dog to infectious and neoplastic diseases. Further, even though

preliminary experiments on immunizing dogs with irradiated larvae have

shown promise (Wong et al., 1974), the results of the present investi-

gation demonstrate that the complexity of the immune response to








D1. immwnnis should be more clearly defined before it can be effectively

manipulated to prevent infections.

One of the most interesting considerationsin view of the depressed

immune response is the possibility of treating the infection with an

agent that will potentiate the immune response in conjunction with,

or beyond, killing the parasite. Levamisole, a compound often used

in immunologic enhancement (Bruley-Rosset, 1976), has been highly

effective in killing D. immitis microfilariae (Bradley, 1976) and

variably effective against adult womns (Boring and Shepard, 1974).

The mode of action of levamisole has not been ascertained, but may be

related to an enhanced immune responsiveness. This principle has been

investigated in the treatment of human filariases (Pinon et al., 1974),

but inconclusive results were obtained. Experiments evaluating the

effectiveness of an immune-enhancing agent against D. irnmitis in dogs

may provide information to aid the treatment of human filariases as

well as canine dirofilariasis.














CONCLUSIONS

Soluble somatic extracts of D. immitis males, females, and

microfilariae were separated into 7, 7, and 3 fractions, respectively.

The isoelectric point range, the number of protein constituents after

PAGE and PAGE-SDS and the estimated molecular weight of each constituent

after PAGE-SDS were determined. A good deal of complexity was present

in most fractions as evidenced by specific protein staining of the

polyacrylamide gels; stains to detect carbohydrate moieties were

unsuccessful. Antigenic activity was demonstrated in each adult D. irritis

fraction and 2 of 3 microfilarial Fractions. Triton X-100 solubilization

of adult D. iimitis cuticles yielded preparations that were weakly

antigenic with few protein constituents.

A TCA-soluble column-purified antigen was demonstrated to be more

effective in the IHA test than previously used antigens. Antibody titers

in experimentally-infected dogs decreased after the appearance of

microfilariae. Titers from dogs infected only once dropped and remained

at low levels following the appearance of microfilariae, but antibody

levels in dogs infected a second time were demonstrable throughout the

study.

n. -iti .ir antigen induced lyiphocyte transfonnation could not be

effected in experimentally-infected dogs. This finding may have been

related to a significantly (p<0.0001) depressed mitogen reactivity of

peripheral blood lymphocytes of D. i eitrs-infected dogs. Quantitation

of nonimmune T-cell rosettes and immunization of dogs with SRBC to assay








T-cell numbers and T-cell function, respectively, did not reveal

any differences in infected and noninfected dogs.
































APPENDICES









APPENDIX I

CDivo -iwia *imitic Microfilariae Counts Over

Weeks After Initial Group B (single infection)
D. i-miti3 Infection K-4 H-6 K-1 H-5


0

32

1100

3500

2500

4400

7100

6400

9000


0

0

0

0

0

0

2

500

500

900

1300

2400

2300

4100


0

0

0

0

0

0

0

1

100

300

1300

2900

5000

2800


0

0

0

0

0

0

100

900

3900

4300

5100

10,200

16,900

20,700


the Experimental Period

Group C (double infection)
H-7 K-2 K-6 H-2


0

0

0

0

0

0

100

1900

2200

3100

5100

8900

9600

24,400


0

0

0

0

0

0

1

700

2300

5300

7000

13,000

20,000

11,900


0

0

0

0

0

1

400

800

900

1200

1600

2600

2400

4100


0

0

0

0

0

0

0

11

500

200

700

4600

4000

3000










Microfilariae Counts (Continued)

Group B (singe infection)
K-4 H-6 K-1 H-5


11 ,800

12,600

12,500

24,000

13,300

15,200

16,500

20,600

16,300

15,200


3400

6100

4200

5600

6400

7300

3700

7500

3700

8200


5800

4200

7500

7600

2900

6800

5900

4800

5000

9600


23,400

23,800

30,900

31,500

20,800

27,800

22,600

36,300

26,800

39,600


Group C (double infection)_
H-7 K-2 K-6 H-2


19,400

27,000

24,900

34,200

26,300

28,600

37,900

29,400

26,000

30,400


29,500

30,200

5900

3600

3700

4000

0

0

0

0


3000

5600

4600

7600

7800

7200

4900

7100

6400

8700


9800

2900

4800

13,100

9800

8300

14,300

22,100

9600

15,900


18,600 4100 3900 32,500 33,600


Weeks After Initial
D. in itis Infection


0 9100 14,411








Appendix II. Polyacrylamcide Gel Electrophoresis in
Sodium Dodecyl Sulfate: Crude Antigen Preparations
and Soluble Somatic Fractions


A. 7.5% polyacrylamide


Cytochrome C
Chynotrypsinogen A
Oval bumin
Bovine serum albumin
Aldolase
Catalase
Crude male
Crude female


C. 12% polyacrylamide

1. Female 2'
2. Female 3'
3. Female 4'
4. Female 5'
5. Female 6'
6. Female 7'
7. Female 8
8. Female 9'
9. Female 10'
10. Female 11'

E. 12% polyacrylamide

1. Male 8'
2. Male 9'
3. Male 10'
4. Male 11 '
5. Microfilaria 1'
6. Microfilaria 2'
7. Microfilaria 3'
8. Microfilaria 4'
9. Microfilaria 5'
10. Microfilaria 6'


B. 12% polyacrylamide

1. Cytochrome C
2. Chymotrypsinogen A
3. Ovalbumin
4. Bovine serum albumin
5. Aldolase
6. Catalase
7. Crude microfilaria
8. Crude male
9. Crude female


D. 12% polyacrylamide

1. Female 12'
2. Female 13'
3. Female 14'
4. Male 1'
5. Male 2'
6. Male 3'
7. Male 4'
8. Male 5'
9. Male 6'
10. Male 7'

F. 10% polyacrylamide


Cytochrome C
Chymotrypsinogen A
Ovalbumin
Bovine Serum albumin
Aldolase
Catalase
Triton X-100 male
Triton X-100 female











A 123456


78


C 12 3 4 5 6 78 910





E .







1 2 34 56 7 8910


B 1 2 34 56 789












D 1 2 3 4 5 6 7 8910


F 1 2 3 4
5


5678


.414-





























Appendix III. Standard Molecular Weight Curve
for 7.5% Polyacrylamide Gel

A= Bovine Serum Albumin
B= Catalase
C= Ovalbumin
D= Aldolase
E= Chymotrypsinogen A
F= Cytochrome C




























0





OLO

w 10
cjLL 00O



LiJ

0

w0




-0
co
F








-0>




T--J
m
0
0
to















0V 01 X)JiH D (Ci no_3


























Appendix IV. Standard Molecular Weight Curve
for 10% Polyacrylamide Gel

A= Bovine serum albumin
B= Catalase
C= Ovalbumin
D= Aldolase
E= Chymotrypsinogen A
F= Cytochrome C




























0






Li 0
d -0
10





oi Lii
Ldd
U)
E



0 ~>-

m
0



-0



100










u~) ~ i- rO C\j-
(tO X i 011M d -lno -lo




























Appendix V. Standard Molecular Weight Curve
for 12% Polyacrylamide Gel

A= Bovine serum albumin
B= Catalase
C= Ovalbumin
D= Aldolase
E= Chymotrypsinogen A
F= Cytochrome C


































(0





0~0
o




clii


0






0
II
cCu
















(0 M 03 Cu


t ol X) IHEDIBM jv-flDo3-ov








Appendix VI. Polyacrylamide Gel Electrophoresis: Crude
Antigen Preparations and Soluble Somatic Fractions

A. 1. Crude male

2. Crude female

3. Crude microfilaria

4. Male 1

5. Male 2

6. Male 3

B. 1. Male 4

2. Male 5

3. Male 6

4. Male 7

5. Female 1

6. Female 2

C. 1. Female 3

2. Female 4

3. Female 5

4. Female 6

5. Female 7

6. Microfilaria 1

0. 1. Microfilaria 2

2. Microfilaria 3

3. Triton X-100 male

4. Triton X-100 female

















A
1 2 3 4 5 6



















C
1 2 3 4 5 6











f^,, .
TrE


3 4 5

;*


B
1 2
D-


















D









APPENDIX VII


Anti-cipofitl!ria imit-i Antibody Titers from All Dogs Over the Experimental Period

Weeks After Initial Group A (noninfected) Group B (single infection) Group C (double infection)
'. ,riitic Infection H-3 K-5 K-3 H-4 K-4 H-6 K-1 H-5 H-7 K-2 K-6 H-2


-4 a 0 1 0 1 0 1 1 1 0 1

-2 1 1 2 1 1 1 1 1 1 1 1 1

0 0 0 0 0 1 1 1 1 1 0

2 0 0 0 0 0 0 0 0 0 0 1 1 0

4 2 1 2 2 4 4 2 4 4 16 8 ?

6 1 0 1 0 2 128 1 2 2 8 1 2

8 2 1 1 1 8 4 8 8 2 64 4 4

10 2 2 1 1 4 4 4 4 2 64 8 2

12 1 2 1 1 4 1 4 2 1 8 8 2

14 2 1 2 2 1 1 2 2 4 32 2 2

16 0 0 1 0 2 0 4 1 2 2 1 1

18 1 1 2 1 32 2 2 2 4 1 1

20 0 0 0 0 128 2 4 8 1 4 8 1

22 0 0 2 0 256 2 2 2 2 4 4 1










Anti-3. iimitis Antibody Titers (Continued)


Weeks After Initial Group A (noninfected) Gr
D. imritiz Infection H-3 K-5 K-3 H-4 K-


1 0

0 0

2 1

1 0

0

0 2

0 1

0 0

1 0


oup B (single infection) Group C (double infection)
-4 H-6 K-1 H-5 H-7 K-2 K-6 H-2


8 1 4 2 1 4 1 1

8 1 1 2 2 16 2 2

1 2 8 2 2 4 2 2

6 1 32 2 4 4 2 1

4 2 64 2 4 16 512 2

8 4 15 4 4 4 2 2

2 4 4 8 4 32 4 4

2 1 32 4 4 8 4 4

2 2 2 2 2 4 4 2

4 1 2 2 4 4 2 2

1 0 1 2 2 4 2 1

2 1 1 2 2 4 2 2

2 1 4 2 2 8 2 4

1 4 16 0 16 1024 4 256

4 1 2 4 4 128 128 64









Anti-D. inrnitis Antibody Titers (Continued)

Weeks After Initial Group A (noninfected) Group B (single infection) Group C (double infection)
D. imritis Infection H-3 K-5 K-3 H-4 K-4 H-6 K-1 H-5 H-7 K-2 K-6 H-2


54 0 1 1 1 2 1 2 4 2 32 8 4

56 0 1 1 1 1 1 2 2 2 16 8 4

58 0 2 2 1 1 1 4 1 1 16 2 1

60 0 2 2 2 1 1 4 2 1 32 2 1

62 0 1 1 1 1 0 2 1 1 16 2 1

64 0 1 1 1 1 0 4 1 1 16 4 2


aAntibody titers are expressed as the reciprocal of the last dilution with hemagglutinating activity.










APPENDIX VIII

Mean Anti-Sheep Red Blood Cell Antibody Titers Before and After
2-Mercaptoethanol Treatment


Group A (noninfected)
H-3 K-5 K-3 H-4


0/0a

32/16

12/6

48/32

32/16

16/16

16/16


0/0

96/96

16/12

64/64

64/48

32/24

64/48


0/0

32/16

12/8

32/16

64/32

32/32

32/32


0/0

16/16

6/4

16/16

16/16

16/16

16/16


Groups B &
K-4 H-6 K-1 H-5


0/0

48/32

16/12

64/32

48/48

48/32

32/32


0/0

24/12

8/6

24/16

32/24

24/16

24/24


0/0

48/32

16/16

64/64

96/48

48/32

64/64


0/0

24/24

6/5

32/24

48/32

32/32

16/24


C (infected)
H-7 K-2 K-6 H-2


0/0 0/0 .5/.5 0/0

32/32 320/128 80/80 24/24

24/16 32/24 24/16 12/6

96/48 128/128 32/32 16/8

64/48 48/32 48/32 16/16

48/32 192/160 48/32 16/8

64/48 256/128 28/32 12/8


aAntibody titers are expressed as the mean of the reciprocal of the last dilution with hemagglutinating
activity.


Days After
Primary
Immunization














APPENDIX IX

Abbreviations Defined and Used in the Text.

B-cell Thymus independent lymphocyte

Con A Concanavalin A

cpm counts per minute

DIA Dirofiaria imnnitis antigen

EDTA Ethylenediaminetetracetic acid

FCS Fetal calf serum

FSSA Female soluble somatic antigen

HAB Hemagglutination buffer

HBSS Hank's balanced salt solution

HRBC Human red blood cells

IgG Irmunoglobulin class G

IgM Immunoglobulin class M

IHA Indirect heiagglutination

MFSSA Microfilaria soluble somatic antigen

MSSA Male soluble somatic antigen

PAGE Polyacrylamide gel electrophoresis

PAGE-SDS Polyacrylamide gel electrophoresis in sodium dodecyl sulfate

PAS Periodic acid Schiff's stain

PBS Phosphate buffered saline

PHA Phytohemagglutinin P

PPD Purified protein derivative-tuberculin antigen

PWM Pokeweed mitogen




Full Text

PAGE 1

CELL-MEDIATED AND HUMORAL IMMUNE RESPONSES TO Dirofilaria irwrltis IN EXPERIMENTALLY-INFECTED DOGS By Robert Burton Grieve A Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy University of Florida 1978

PAGE 2

ACKNOWLEDGEMENTS I extend my gratitude to Dr. Richard Bradley, supervisory chairman, for supporting this research and for the countless lessons he taught. The suggestions and guidance of the supervisory committee, Drs. P. T. Cardeilhac, H. L. Cromroy, and E. M. Hoffmann, are appreciated. Dr. M. D. Young participated in each committee meeting and offered help and suggestions whenever needed. The generosity and guidance of Dr. John Neil son is gratefully acknowledged. I was especially fortunate to benefit from his experience and special abilities in helminth antigen isolation and characterization. The insight obtained with his help will be invaluable in future research and was principally responsible for obtaining an ideal postdoctoral posi tion. Dr. Bryan Gebhardt helped in so many ways they cannot all be listed. He provided boundless time, patience, and expertise in all the aspects of the immune response researched in this dissertation. He frequently offered helpful criticism and suggestions and further, he provided a good deal of encouragement at times when it helped. His assistance is greatly appreciated. There have been valued associations within and outside the laboratory with fellow graduate students. Dann Brown, Nguyen Van Dat, Carlos Costa, John Kennedy, Dan Murfin, and Wallace Randell have been remarkable friends whose special and diverse interests in parasitology contributed to an interesting and beneficial research atmosphere. Each of them

PAGE 3

offered time and suggestions essential to the completion of this work. Mr. Dat deserves additional thanks for all his help on the statistical analyses in this investigation. The assistance of Dr. H. Neil Becker in providing veterinary services critical to various aspects of this research is appreciated. Special thanks are due Mr. Joe DiCarlo who provided outstanding technical assistance in Dr. Gebhardt's laboratory. The assistance of Mr. Louis Ergle in coordinating technical help in Dr. Bradley's laboratory is appreciated. The patience and typing skill of Ms. Dianna Jackson is sincerely appreciated. This dissertation would have been much slower developing without her kind help. The support of all my family is sincerely acknowledged. It is not possible to describe the many ways they have all helped. However, a very special acknowledgement is in order for Jane, my wife. She left a career she enjoyed to move to Florida and has worked at three mediocre jobs in a town where student wives are both underappreciated and underpaid. Jane somehow managed a budget and household with very meager funds, accepted the long, late hours at the laboratory, encouraged me through the many rigors of graduate study, and somehow found time to develop several independent interests. She has my deepest respect and appreciation for all these things and more. A graduate assi stantship was provided by the College of Veterinary Medicine (Institute of Food and Agricultural Sciences Animal Research Facility). The research was supported in part by Hatch Project 1419 (W-102). 1 1 1

PAGE 4

TABLE OF CONTENTS Page ACKNOWLEDGEMENTS i i LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT viii INTRODUCTION 1 LITERATURE REVIEW 4 MATERIALS AND METHODS 12 Experimental Animals 12 Experimental Design 12 Experimental Infections 13 Blood Collections 13 Antigen Purification and Characterization 14 Protein Determinations 14 Crude Antigen Extraction 14 Preparative Isoelectric Focusing 16 Production of Rabbit Antisera 17 Immunodiffusion 18 Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate 20 Polyacrylamide Gel Electrophoresis 21 Humoral Immune Response Determinations 22 Purification of the Indirect Hemagglutination Antigen 22 Indirect Hemagglutination Assay 26 Cell-Mediated Immune Response Determinations 29 Lymphocyte Isolation 29 Lymphocyte Transformation 30 Lymphocyte Rosette Assay 32 Immune Response to Sheep Red Blood Cells 32 RESULTS 34 Microfilariae Counts 34 Antigen Purification and Characterization 34 Preparative Isoelectric Focusing 34 Immunodiffusion 37 Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate 37

PAGE 5

Page Polyacryl amide Gel Electrophoresis 47 Humoral Immune Response 47 Cell -Mediated Immune Response 50 Lymphocyte Transformation 50 Lymphocyte Rosettes 50 Immune Response to Sheep Red Blood Cells 50 DISCUSSION 60 Antigen Purification and Characterization 60 Humoral Immune Response 63 Cell -Mediated Immune Response 65 CONCLUSIONS 71 APPENDICES I. Dirofilaria Lmmitis Microfilariae Counts Over the Experimental Period 74 II. Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate: Crude Antigen Preparations and Soluble Somatic Fractions 77 III. Standard Molecular Weight Curve for 7.5% Polyacrylamide Gel 79 IV. Standard Molecular Weight Curve for 10% Polyacrylamide Gel 81 V. Standard Molecular Weight Curve for 12% Polyacrylamide Gel 83 VI. Polyacrylamide Gel Electrophoresis: Crude Antigen Preparations and Soluble Somatic Fractions .... 85 VII. AntiDirofilaria immitis Antibody Titers from All Dogs Over the Experimental Period 86 VIII. Mean Anti -Sheep Red Blood Cell Antibody Titers Before and After 2-mercaptoethanol Treatment ... 89 IX. Abbreviations Defined and Used in the Text 90 BIBLIOGRAPHY 92 BIOGRAPHICAL SKETCH 99

PAGE 6

LIST OF TABLES Table 'age 1. Divnfilaria immitis Male Soluble Somatic Antigen Fractions Purified By Preparative Isoelectric Focusing and Characterized By Immunodiffusion, Polyacrylamide Gel Electrophoresis, and Polyacry1 amide Gel Electrophoresis in Sodium Dodecyl Sulfate 44 2. Divofilaria immitis Female Soluble Somatic Antigen Fractions Purified By Preparative Isoelectric Focusing and Characterized By Immunodiffusion, Polyacrylamide Gel Electrophoresis, and Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate 45 3. Divofilaria immitis Microfilaria Soluble Somatic Antigen Fractions Purified By Preparative Isoelectric Focusing and Characterized By Immunodiffusion, Polyacrylamide Gel Electrophoresis, and Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate 46 4. Mean Counts Per Minute and Stimulation Indices From Four Canine Peripheral Blood Lymphocyte Transformation Experiments of Dirofilaria immi tic-Infected Dogs and Noninfected Dogs 51 5. Percentage of Canine Peripheral Lymphocytes Forming Nonimmune Rosettes with Human Erythrocytes .... 52

PAGE 7

LIST OF FIGURES Figure Page 1. Diagrammatic Representation of the Scheme Used in Purification and Characterization of Divofilavia immitis Antigens 24 2. Diagrammatic Representation of the Procedure for Purification of the Divofilaria immitis Indirect Hemagglutination Antigen 27 3. Mean Numbers (XI ) of Divofilaria immitis Microfilariae Per ml of Blood in Group B (single infection) and Group C (double infection) 36 4. Preparative Isoelectric Focusing Separation and Percentage of Recovered Protein in the Final Fractions from the divofilaria immitis Male Soluble Somatic Extract .... 39 5. Preparative Isoelectric Focusing Separation and Percentage of Recovered Protein in the Final Fractions from the Divofilaria immitis Female Soluble Somatic Extract ... 41 6. Preparative Isoelectric Focusing Separation and Percentage of Recovered Protein in the Final Fractions from the Divofilaria immitis Microfilaria Soluble Somatic Extract 43 7. Mean log 2 of the Reciprocal Anti -Divofilaria immitis Antibody Ttters in Groups A, B, and C Over the Course of Infection 49 8. Mean log„ of the Anti-Sheep Red Blood Cell Antibody Titers in Group A anc' Groups B + C 54 9. Mean Difference of log 2 of Reciprocal Anti-Sheep Red Blood Cell Antibody Titers Before and After Serum Treatment with 2-mercaptoethanol 56 10. Separation Profile of Pooled Canine Anti -Sheep Red Blood Cell Serum on Sepharacyl S-200 and the Pools Collected for 2-mercaptoethanol Lability Determinations 59

PAGE 8

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CELL-MEDIATED AND HUMORAL IMMUNE RESPONSES TO Dirofilaria immitis IN EXPERIMENTALLY-INFECTED DOGS By Robert Burton Grieve March 1978 Chairman: Richard E. Bradley Major Department: Animal Science The canine immune response to Dirofilaria immitis was investigated in 3 aspects. Antigens from D. immitis adults and microfilariae were isolated and partially characterized and the humoral and cell-mediated immune responses to D. immitis were studied in experimentally-infected dogs. Antigens from soluble somatic extracts of D. immitis males, females, and microfilariae were separated using preparative isoelectric focusing. The fractions obtained by isoelectric focusing were characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (PAGE-SDS) and by immunodiffusion against canine sera from D. immitis -infected dogs and homologous rabbit antisera. Adjacent fractions from each preparation were combined on the basis of isoelectric point, similarity of protein constituents, and antigenic identity. The combined fractions were further characterized by polyacrylamide gel electrophoresis (PAGE). Gels were stained with coomassie blue for protein constituents and periodic acid

PAGE 9

Schiff ' s stain and alcian blue were used to detect carbohydrate moieties. Triton X-100 was used to solubilize cuticle-bound antigens from D. immitis males and females; the resultant preparations were characterized by the same scheme employed for the soluble somatic fractions. Soluble somatic preparations of males, females, and microfilariae were divided into 7, 7, and 3 final fractions, respectively. Considerable complexity and a wide molecular weight range of constituent proteins were observed in most fractions. All male and female fractions and 2 of 3 microfilaria fractions reacted with canine sera from D. immitisinfected dogs and homologous rabbit antisera. In contrast, the Triton X-100 solubilized preparations were less complex and demonstrated weak reactivity against canine sera from D. trawls -infected dogs and rabbit antisera. Both stains for carbohydrate determinations after PAGE were unsuccessful with the soluble somatic fractions. A trichloroacetic acid-soluble, column-purified D. immitis antigen was used in an indirect hemagglutination assay to determine anti-O. inmitis antibody titers in experimentally-infected dogs. Antibody titers were determined in 4 dogs with a single u. immitis infection, 4 dogs with a second D. immitis infection administered after the maturation of the first infection, and in 4 uninfected dogs. Sera for antibody determinations were collected every 14 days beginning 4 weeks before infection until 64 weeks after infection. Anti-/). immitis antibody was first detected in infected dogs 4 weeks after infection. Titers were highest at week 32, 2 weeks after the appearance of microfilariae, and diminished to low levels thereafter in the single infection group. IX

PAGE 10

Antibody levels in the double infection group diminished as in the single infection group, but were demonstrable throughout the study. Antibody titers were higher (p<0.05) in infected dogs, but there were no significant differences in antibody titers between single and double infection groups. The cell-mediated immune response was investigated using peripheral blood lymphocyte transformation. Lymphocyte transformation could not be induced with D. immitis antigens and the response to the mitogens phytohemagglutinin P, pokeweed mitogen, and concanavalin A were significantly depressed (p<0.0001) in infected dogs. Efforts to quantitate thymus dependent lymphocytes (T-cells) using nonimmune T-cell rosette formation with human red blood cells revealed no differences in T-cell numbers in infected and noninfected dogs. All dogs (infected and noninfected) were immunized with a T-cell dependent antigen, sheep red blood cells (SRBC), to investigate possible differences in T-cell functions in infected and noninfected dogs. No differences between groups in anti-SRBC antibody levels or 2-mercaptoethanol labile antiSRBC antibody levels could be demonstrated.

PAGE 11

INTRODUCTION The canine heartwomi, Divofilaria immitis, has been the subject of considerable research. A more complete understanding of this infection is important both from the standpoint of the severity of the disease it may cause and possible ways to control the growing incidence of the parasite (Otto, 1974a). Further, the parasite often serves as a model for human filariasis research and may offer insights into the control of those infections. Research on methods to control D. immitis has been substantial but has been directed mainly to chemoprophylaxis and chemotherapy. Although chemoprophylaxis is reliable, it is not totally effective (Grieve and Bradley, unpublished information) and can be costly and inconvenient. Treatment to kill adult D. immitis is hazardous because of the danger of drug toxicity and the potential damage of emboli from dead parasites associated with the arsenical compound currently employed (Carlisle et at. , 1974). As an alternative to chemotherapy, research on biologically-derived antigens to be used as vaccines has been emphasized in recent years, but it has become evident that before effective immunologic methods for the prevention or diagnosis of such infections can be developed, the responses of the host to the parasite and the mechanisms by which the parasite survives in an otherwise immunologically competent, natural host must be elucidated. The canine immune response to D. immitis has been the topic of several investigations, but these studies have often relied upon noncontrolled, natural infections or limited animal

PAGE 12

numbers. In the few reports of experiments that utilized experimental infections, crude, nonspecific antigens were used which may have limited the amount of information produced. When the data reported from previous investigations on d. imrnitis immunity are considered, a number of questions become apparent. In an effort to answer some of the questions, three major objectives were developed for this dissertation. The three major objectives addressed in this research were: 1. Isolation and partial characterization of antigens of u. imnitis males, females, and microfilariae. Rationale: Since different life-stages are present in the dog during the course of infection, information on the actual antigenic constituents and antigenic relationships of different stages (adult males, adult females, and microfilariae) is central to understanding the host immune response. Information on the complexity of the antigenic constituents of these stages is prerequisite to further study of possible genusspecific or stage-specific antigens. 2. Elucidation of the humoral immune response in dogs following primary and secondary experimental infections with d. imnitis. Rationale: In all previous reports concerning the humoral response in experimentally-infected dogs a crude, nonspecific antigen was used (Pacheco, 1966; Weiner and Bradley, 1972). Since a highly specific and sensitive adult worm antigen has been reported (Mantovani and Kagan, 1967), it was considered advantageous to use that antigen to study the antibody response in experimentally-infected dogs. 3. Investigation of the cell-mediated immune response to D. imnitis in experimentally-infected dogs using the in vitro lymphocyte transformation technique.

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Rationale: Assays of the cell mediated immune response to D. immitis have been either biological (Mantovani and Kagan, 1967) or have measured the in vitro response to a D. immitis antigen in an unnatural host (Kobayakawa, 1975). Examination of the response of lymphocytes from D. immitisinfected dogs to D. immitis antigens may provide new information on the nature of the immune response and on possible mechanisms of parasite survival. The experimental design of this investigation was intended to help gain new, fundamental information on the canine immune response to D. immitis infections under controlled conditions. The intent of this study was to help in better understanding the infection and to explore the possibilities of any practical applications of that response.

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LITERATURE REVIEW Bivofilavia immitis Leidy, 1856, the canine hear two rm, is distributed throughout the world; it is especially prevalent in warm, coastal regions and notably rare in Africa (Levine, 1968). In the United States, the infection appears to be extending from the enzootic Atlantic and Gulf Coast regions to the north and west and occurs in highly enzootic rates in certain temperate areas (Otto, 1974a) The nature habitat of adult P. immitis is in the right ventricle and adjacent vasculature of the dog, Canis familiaris. However, the parasite has also been recorded as recovered from vomitus, feces, the eye, brain, spinal cord, abdominal cavity, bronchioles, various arteries, and abcesses (Otto, 1974b). Wild canines and felines may also serve as definitive hosts (Otto, 1974b) and a number of naturally-infected abnormal hosts have been reported including the black bear (Johnson, 1975), beaver (Foil and Orihel, 1975), wolverine (Williams and Dade, 1976), harbor seal (Medway and Wieland, 1975), California sea lion (Forrester at at., 1973), domestic horse (Klein and Stoddard, 1977), and human (Otto, 1974b). Experimental infections have been used to characterize the pathogenesis of D. immitis infection in felines (Donahue, 1975). Adult worm recoveries from experimentally infected cats are low, and dead worms found in many cats indicate that the dog is a more suitable host. Macaques have been experimentally infected to study P. immitis infections of primates (Wong, 1974). Parasites reached maturity only in those

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macaques that were chemically immunosuppressed; it was concluded that a period of 60-90 days of diminished immune responsiveness after infection is necessary for the worms to reach maturity in the heart. Adult D. immitis live preferentially in the right ventricle and pulmonary arteries of dogs. Adult males are 120-200 mm long and 100900 ij wide with a spirally coiled tail characteristic of most f i 1 arid males; females are 250-310 mm long and 1.0-1.3 mm wide (Levine, 1968). Microfilariae, the first-stage larvae, develop in the uterus of the adult female worm and are expelled without a sheath, gut, or genital primordium. The microfilariae are 286-340 y long and 6.1-7.2 y wide with a tapered anterior end and a straight tail (Lindsey, 1965). Microfilariae are present in the peripheral circulation of dogs with mature infections and have displayed seasonal (Kume, 1974) and daily periodicity (Pacheco, 1974). The mechanisms for periodicity are unknown (Masuya, 1976) but increased numbers of microfilariae in peripheral circulation during periods of greater vector availability facilitates perpetuation of the infection. A relatively constant number of microfilariae remain in the circulation of any given infected dog. The mechanism for maintaining a stable microfilariae population is unknown and withdrawal or addition of large numbers of microfilariae will not alter the population present in peripheral circulation (Wong, 1964). Several mosquito species have been identified as proven D. immitis vectors (Levine, 1968). Microfilariae are ingested by the mosquito at the time of the blood meal and undergo two molts within the mosquito before they are infective as third-stage larvae. Third-stage larvae enter the dog where the mosquito has fed and molt in subcutaneous tissue to fourth-stage larvae in 9-12 days. A subsequent molt to fifth-stage

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larvae occurs 60-70 days after infection (Orihel, 1961). A microfilaremia indicating maturity is usually evident at 6.5 months (Orihel, 1961). D. immitis infections of dogs are typically diagnosed by demonstrating the characteristic microfilariae in the blood. However, no real correlation can be made between numbers of microfilariae and numbers of adult worms (Jackson, 1969; Pacheco, 1974). This method of diagnosis is further complicated by situations where adult worms are present without circulating microfilariae. Single sex infections or infections in an animal that will respond in a unique immunological fashion to microfilariae have no circulating microfilariae (Wong et al., 1973). Serious disease may result from a long-standing infection with D. immitis, especially if a large number of worms are present in the dog's circulatory system. Adult worms may produce a chronic endocarditis and dilation of the right heart; lungs may be congested and are typically effected by the granulomatous responses to thrombi formed from dead worms (Levine, 1968). Perhaps the most important pathology in the lungs is the resultant endarteritis and obstructive fibrosis (Adcock, 1961). In chronic cases, the liver may become enlarged and ascites will ensue. A terminal liver failure syndrome has been described in experimentally-infected dogs (Sawyer and Weinstein, 1963). Glomerular changes have also been reported in infected dogs. Although an immune complex associated etiology has been postulated (Casey &t al., 1972), evidence indicates that lesions are related to damage caused by motile microfilariae (Klei at al., 1974; Simpson el al., 1974). Most reports indicate that there is not a protective immune response to D. immitis . Adult heartworms are patent for at least two

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years (Levine, 1968) and microfilariae may persist when transfused into uninfected dogs for as long as two years (Underwood and Harwood, 1939). Under natural conditions in enzootic areas, dogs may be repeatedly infected over a lifetime, but after subjective analysis of such reports, it appears that the infection levels are not additive. There is experimental evidence that dogs can be successfully immunized against D. immitis with irradiated D. immitis infective larvae (Wong et at., 1974). Dogs that received varying numbers of gammairradiated third-stage larvae injected on different schedules revealed up to 0% recovery of adult worms after challenge with nonirradiated larvae. Antibody levels were not high and there was no anamnestic response after repeated challenge with either irradiated or nonirradiated larvae. Since D. immitis is closely related to many of the human filarids it is often used as a source of antigen in the immunologic diagnosis of human filarial infections. Cross-reacting antigens among several filarids have been demonstrated (Neppert, 1974) and the availability of D. immitis makes it a good antigen source. Early antigenic preparations for diagnostic use were typically overnight saline extracts of macerated microfilariae (Franks and Stoll, 1945) or adults (Kagan, 1963). Presently, saline extracts of lyophilized adult D. immitis are used to obtain antigen preparations for the serodiagnosis of human filariases at the National Communicable Disease Center (Kagan and Norman, 1976). Indirect hemagglutination and bentonite flocculation tests employing this antigen have been relatively sensitive, but cross-reactivity is not restricted to filarids. Unfortunately, cross-reactions with the sera of patients infected with other nematodes, cestodes, trematodes,

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protozoans, and even bacteria have been reported (Kagan and Norman, 1976). Pacheco (1966) reported on the sensitivity and specificity of different D. immitis whole worm extracts. In this study, a saline extract of lyophilized worms, a del ipidized saline extract of lyophilized worms, acid-insoluble and acid-soluble protein extracts, and an ethanol extract were compared. The acid-insoluble fraction and the ethanol extract did not react with known positive anti-D. inmitis canine antisera using indirect hemagglutination. The acid-soluble protein preparation was the most specific and sensitive of the antigen preparations tested, but sera of some animals infected with other helminths often crossreacted. Fluorescent antibody techniques have also been used with crude antigen preparations of adults (Ellsworth and Johnson, 1973; Wong, 1974), microfilariae (Wong, 1974; Quails et al., 1975), and third-stage larvae (Wong, 1974). Wong (1974) reported the ability to discern stagespecific antibody using an indirect fluorescent antibody technique with crude adult, microfilaria! and larval antigens. The initial, extensive purification procedures of D. inmitis antigens were reported in 1965 (Sawada et al., 1965). That work was designed to isolate filarial skin test antigen to diagnose human filarial infections. An aqueous, soluble somatic antigen preparation from adult D. immitis was treated with trichloroacetic acid and purified by gel filtration and ion-exchange chromatography. A fraction demonstrating a good degree of specificity and sensitivity was isolated and has been proven to be of benefit (Smith, 1971). In later work (Sawada et al., 1970), anion-exchange chromatography, disc electrophoresis, and isoelectric focusing were employed to show at least 17 different proteins

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in the isolated fraction. The various subtractions obtained were variably reactive in the skin test and were still relatively complex. The isolation and purification scheme of Sawada et at. (T965) was repeated to obtain a highly specific and sensitive antigen fraction for use in the diagnosis of canine heartworm infection (Mantovani and Kagan, 1967). This antigen was reactive in skin tests and by the indirect hemagglutination assay it was shown to be specific for D. immitis when tested in dogs naturally infected with D. hnnitis, D. re-pens, and Dipetalonema recondition. The advantage of the genus and species specificity of this antigen would be unique but, unfortunately, it has not been used in studies on the kinetics of the canine humoral immune response to D. immitis. Another study reported that aqueous soluble extracts of D. immitis adults and microfilariae were subjected to disc electrophoresis (Wheeling and Hutchison, 1971). This technique revealed at least 27 and 17 protein bands, respectively. Immunoelectrophoresis of the adult extract against homologous rabbit antisera showed 10 precipitin arcs; 2 arcs were noted against sera from a patient with suspected filariasis. One arc developed when the microfilariae extract was subjected to Immunoelectrophoresis against the human sera. Takahashi and Sato (1976) used a defatted aqueous-soluble extract of adult D. immitis for fractionation. A fraction that was very reactive and specific in serodiagnosis of Wuchereria bancroftiinfected patients was reported after gel filtration, anion-exchange chromatography, and ammonium sulfate precipitation. Disc electrophoresis showed at least five protein bands with activity from the purified fraction.

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More recently countercurrent Immunoelectrophoresis of u. immitis male and microfilaria! aqueous soluble somatic antigens against homologous rabbit antisera was used to demonstrate apparent stage-specific antigens (Desowitz and Una, 1976). Additionally, this same technique was used with sera from hypermicrofilaremic dogs and rabbit anti-P. immitis antisera to demonstrate circulating soluble D. immitis antigens. Sera from humans with low microfilaremias or occult infections showed intense precipitin lines to microfilaria-specific antigens. The humoral immune responses to experimental D. immitis infections in dogs were studied in long term experiments with experimental infections by 2 investigators (Pacheco, 1966; Weiner and Bradley, 1972). In one study, anti-D. iinmitis antibodies were detected by indirect hemagglutination as early as two weeks after experimental infection, but could not be detected by either indirect hemagglutination or complement fixation beyong the ninth month postinfection (Pacheco, 1966). Peaks of antibody titers were observed at different times over the course of the prepatent and patent periods. It was postulated that they may have been related to antigenic changes in the parasite or to different antibody class responses. The author suggested that the lack of detectable antibody shortly after patency was due to absorption of most of the antibody by circulating microfilariae. Weiner and Bradley (1972) investigated the humoral immune response after primary and secondary experimental D. immitis infections. A crude aqueous-soluble adult worm antigen in an indirect hemagglutination assay was used and serologic responses were similar to those reported by Pacheco (1966). A peak titer appeared about 22 weeks after infection and the amount of detectable antibody declined subsequent to the

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appearance of microfilariae. Secondary infections administered after patency of the first infections did not produce an anamnestic response, but did delay the decrease in antibody titers as had been observed in the dogs infected only once. In further work, Weiner and Bradley (1973) used single, radial immunodiffusion and 2-mercaptoethanol lability to demonstrate that immunoglobulin class M (IgM) was probably the primary active immunoglobulin throughout D. immitis infections. They speculated that immunoglobulin class G (IgG) may also have some role, but it was not demonstrable by the techniques used. To date, most assays of the eel 1 -mediated immune response have relied on skin testing. Mantovani and Kagan (1967) used the skin test to evaluate the column-purified antigen. The results obtained supported the serologic observations of D. immitis specificity. Kobayakawa (1975) reported on extensive research on the guinea pig eel 1 -mediated immune response to a defatted adult D. immitis extract. A response was confirmed by the migration inhibition test, the 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. However, in vitro assays of the canine cellmediated immune response to D. immitis have not been reported.

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MATERIALS AND METHODS Experimental Animals Twelve pedigreed beagle dogs aged 9-10 weeks were obtained from a commercial supplier. Immediately upon receipt the dogs were housed in a Rockefellertype isolation building provided with temperature control and double-screened windows. Water and a commerically available 2 feed were provided ad libitum. Specific procedures were observed to maintain the rooms insect-free and the dogs helminth-free. The dogs were from 2 litters with 3 males and 3 females from each litter; throughout the experiment the animals were divided by sex. Rectal temperatures were determined daily and fecal analyses (Whitlock, 1948) were performed weekly on each dog for 12 weeks after arrival. Fecal analyses of all dogs were performed intermittently throughout the investigation to insure a helminth-free status. Previous to experimentation all dogs were subjected to a standard puppyhood vaccination regimen against canine distemper, canine infectious hepatitis, and leptospirosis. Experimental Desig n The dogs were divided into 3 groups of 4 animals each with 1 male and 1 female of each litter in each group. One group was an uninfected control group (Group A), the second (Group B) and third group (Group C) Hazel ton-Saunders, Inc., Midlothian, VA 2 Gaines Meal, General Foods Corp., White Plains, NY 12

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received single and double D. immitis infections, respectively. Experimental Infections The initial D. immitis infection was administered when the dogs were aged 10 months (Week 0). Approximately 500 female black-eye Liverpool Aedes aegypti that were fed on D. immitis mi crofi Uremic canine blood were obtained from the College of Veterinary Medicine, University of Georgia, Athens. Fifteen days after the infected blood meal the mosquitoes were dissected in Hank's balanced salt solution (HBSS)(pH 7.2). Infective larvae were individually counted into 1 ml disposable syringes; 2 after 30 larvae were counted into a syringe, it was filled to 1 ml with HBSS for inoculation. The larval inoculum was administered subcutaneously in the inguinal region of each dog in Groups B and C, then the syringes were filled with HBSS and the wash was inoculated subcutaneously, opposite the initial inoculation. Each syringe was carefully washed and the wash was examined microscopically for remaining larvae. Thirty-six weeks after the initial infection a second infection of the same larval number was administered to Group C. B lood Collections Blood was collected from the cephalic vein of each dog every 14 days beginning 4 weeks before experimental infection until 64 weeks after infection. Approximately 10ml of blood was collected at each sampling; 3 2 ml were aspirated into a evacuated tube containing EDTA and the Provided by the U.S. -Japan Cooperative Medical Science Program (NIAID) 2 Stylex R , Pharmaseal Laboratories, Glendale, CA 3 Vacutainer R , Becton, Dickinson and Co., Rutherford, NJ

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remainder was used for individual serum samples. Serum collected from whole blood was stored at -35°C for anti-D. immitis antibody determinations. Anticoagulant treated blood was used to count microfilariae (Weiner and Bradley, 1970) 2 and 4 weeks before infection, 2 weeks after infection, and then every 14 days beginning 22 weeks after infection. Antigen Purification and Characterization Protein Determinations A standard curve was used to determine protein concentrations of all antigen preparations. A known quantity of bovine serum albumin was serially diluted and the absorbance of each dilution was measured at 280 2 nm using a dual beam spectrophotometer. This provided a standard curve so the absorbance of the various antigen preparations could be related to a protein concentration. Crude Antigen Extraction Adult D. immitis males and females were collected at necropsy from experimentally-infected dogs. The worms were separated by sex, washed three times in phosphate buffered saline (PBS)(pH 7.2), and frozen at -70°C. Microfilariae were collected by immersing female D. immitis recovered from naturally-infected dogs into PBS (pH 7.2) at 4°C for 2 hours; the adults were removed and the microfilariae were recovered 3 after centrifuging the PBS at 12,100 X g for 15 minutes at 4°C. Microfilariae were washed twice in PBS and frozen at -70°C. American Monitor Corp., Indianapolis, IN 2 Unicam SP1800 Ultraviolet Spectrophotometer, Philips Electronic Instruments, Mount Vernon, NY Beckman J-21C Centrifuge, Beckman Instruments, Inc., Palo Alto, CA

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Each whole worm preparation was minced with a razor blade and mixed with approximately 5 times the worm volume of cold 0.01M 2-amino2(hydroxymethyl )-l ,3-propanediol (tris)(pH 8). The mixture was further disrupted using a french pressure cell press at 2.0 X 10 4 pounds per square inch pressure. The homogenate was extracted overnight at 4°C on 2 a continuous rocker and then centrifuged at 12,100 X g at 4°C for 30 minutes; the supernatant was harvested and the precipitate resuspended in 10 volumes of 0.01M tris (pH 8) and further extracted for 72 hours. The combined supernatants served as the crude soluble somatic extract; the precipitate was extracted with PBS (pH 7.2) until protein was no longer evident and then subjected to detergent extraction. 3 Triton X-100 was used to solubilize any cuticle-associated proteins by the method of Harris et at. (1971). Only male and female preparations were in sufficient quantity for this extraction. The proteinextracted cuticular debris was lyophilized overnight and added to 13% Triton X-100 (pH 7.5) at a rate of 0.1 g/ml and extracted by continuous agitation at 4°C for 72 hours. The mixture was centrifuged at 12,100 X g at 4°C for 30 minutes and proteins were obtained by adding 10 volumes of acetone to the supernatant at -35°C. The precipitate was collected after 24 hours, washed with ether, dried in vacuo and extracted for 24 hours with 0.01M tris (pH 8). The solubilized preparation was dialyzed 1 R Aminco French Pressure Cell, American Instrument Co., Silver Springs, 2 R Lab-Tek Aliquot Mixer, Ames Lab-Tek, Inc., Westmont, IL 3 R Triton X-100, Fisher Scientific Co., Fair Lawn, NJ 4 Labconco Freeze Dry-5, Labconco Corp., Kansas City, M0

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TIT against distilled water with 0.1°/ sucrose at 4°C for 48 hours and lyophilized. The Triton X-100 solubilized fractions of male and female cuticles are referred to as TSM and TSF, respectively. Preparative Isoelectric Focusing Approximately 100 mg of crude male soluble somatic antigen (MSSA), 120 mg of crude female soluble somatic antigen (FSSA), and 80 mg of crude microfilaria soluble somatic antigen (MFSSA) were individually subjected to preparative, flatbed isoelectric focusing. The methodology observed is a modification of the techniques described by Winter et at., (1975). Each crude preparation was dialyzed overnight against l/o glycine at 4°C. A slurry consisted of the aqueous antigen preparation, 2 3 5 g of Sephadex G-75 superfine, 5.0 ml of ampholines pH 3.5-10 ,and distilled water to 100 ml was used to prepare each gel bed. The slurry was mixed, degassed and poured into a glass tray, then evaporated to reach 75% of the slurry crack-point. The gel bed was subjected to a continuous electric current at approximately 7 watts of constant 4 5 power at 10°C for 14-16 hours. After focusing was complete, the current was discontinued and a dry filter paper (5 X 25 cm) was placed over the length of the gel bed for 2 minutes. The paper was removed and a fractionating grid was pressed through the gel bed. The filter paper was air dried, washed 3 times for 15 minutes each time in 10% ] LKB 2117 Multiphor, LKB Instruments, Inc., Rockville, MD 2 Pharmacia Fine Chemicals, Pharmacia, Inc. , Piscataway, NJ 3 R Ampholine pH 3.5-10 LKB Instruments, Inc., Rockville, MD 4 Electrophoresis Power Supply, ISC0, Lincoln, NB 5 Lauda K-4 R/D Brinkman Instruments, Messergate-Werk, Lauda, W. Germany

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trichloroacetic acid (TCA), and slained with 0.2% coomassie brilliant blue dissolved in methanol, water, and acetic acid (50:50:10). After staining was complete the paper was destained with methanol, water, and acetic acid in the same proportions until the background color was absent. The pH of the gel bed at each of the 30 divisions created by the 2 fractionating grid was determined; this data and the staining patterns of the corresponding filter paper print were used to divide the gel bed into distinct fractions. When the constituent divisions of each fraction were determined, the gel bed within those divisions was harvested and washed into glass columns (1 X 30 cm). The columns were eluted with distilled water and 5 ml volumes were collected until protein was not detectable. Total protein in each fraction was determined, then the fractions were dialyzed to remove ampholines, lyophil ized, and solubilized with distilled water to a final protein concentration of 3 mg/ml . If less than 3 nig of protein was present the fraction was solubilized with 1 ml distilled water. Production of Rabbit Antisera Antisera for use in immunodiffusion was made in mature, female New Zealand white rabbits by intradermal inoculations (Vaitukaitus et al., 1971) of crude soluble somatic or protein-extracted cuticle debris preparations. One nig of protein of male, female, and microfilaria! crude Searle Diagnostic, High Wycombe, Bucks-England 2 Corning Glass Works, Corning, NY 3 Calbiochem, La Jolla, CA

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soluble somatic antigens and 1 nig dryweight of male and female cuticular debris were administered to individual rabbits in a suspension of 1 ml of 0.01M tris (pH 3) and 1 ml of complete Freund's adjuvant. The suspension was homogenized by passing it through a 20 gauge micro2 emulsifying needle, then inoculated intradermal ly at 25-30 sites on the back of each animal. At a separate site each animal received 0.5 ml of a vaccine containing diptheria and tetanus toxoids and a 3 pertussis immunogen. Animals were reimmunized by the same procedure 8 days later using Freund's incomplete adjuvant; the vaccine was not used at reimmunization. Twenty-nine days after the second immunization each rabbit received 1 nig of homologous crude antigen by intravenous administration in a marginal ear vein. An adjuvant-control rabbit received the same regimen without crude antigen. Blood was collected from all rabbits by marginal ear-vein puncture beginning 9 days after secondary immunization and then every 3-4 days for approximately 7 weeks. Sera collected from individual rabbits was pooled. Im munodiffusion The pooled rabbit antisera and pooled canine sera from dogs naturally-infected with D. immitis were used in immunodiffusion assays to compare adjacent fractions from preparative isoelectric focusing. The immunoglobulin portion of each pool was isolated by an ammonium sulfate precipitation technique (Harboe and Ingild, 1973). Twentyfive g of ammonium sulfate was added to 100 ml of each serum pool and Calbiochem, La Jolla, CA 2 Bolab, Inc. , Derry, NH 3 R Wyeth , Wyeth Laboratories, Inc. , Marietta, PA

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this mixture was incubated for 20 hours at 22°C. After centrifugation at 12,100 X g for 30 minutes the supernatant was discarded and the precipitate was washed twice in 1.75M ammonium sulfate. The washed precipitate was solubilized with distilled water and dialyzed for 24 hours against distilled water and 24 hours against PBS (pH 7.2). Adjacent fractions collected from isoelectric focusing were compared for antigenic activity and identity against the immunoglobulin preparations of homologous rabbit antisera and canine immune sera using a standard immunodiffusion technique (Anonymous, 1968). Agarose, sodium chloride and distilled water were mixed in the proportions 1:1:98,. and heated to 90°C in a boiling water bath. Twelve ml of the hot solution was poured onto a level glass plate (9X9 cm), then after 2 hours at 2 4°C holes were cut in the solidified gel and 10 nl of each fraction described above was diffused against 10 pi of purified immunoglobulin for 72 hours. Nonprecipi tating protein was washed from the agarose with 1% sodium chloride for 48 hours, then the plates were rinsed in distilled water for 6 hours and dried. Staining was with 0.]% napthyl blue-black in methanol, glacial acetic acid, and water (45:10:45); destaining was in the same solvent. Adjacent fractions showing antigenic identity were combined. TSM and TSF were diffused against homologous rabbit antibody and canine antibody to determine antigenic activity and complexity. Biorad Laboratories, Richmond, CA 2 Gelman Instrument Co., Ann Arbor, MI 3 Sigma Chemical Co., St. Louis, M0

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Polyacrylamide Gel Electro phoresis in Sodium Dodecyl Sulfate Fractions combined after immunodiffusion were subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (PAGE-SDS) to determine the complexity of each fraction and the approximate molecular weight of the fraction constituents. The principle methodology observed was reported by Weber and Osborn (1969) with adaptations for slab PAGE-SDS (Easterday et at, 1976). Fractions combined after immunodiffusion were dialyzed for 72 hours at 4°C against 0.001M tris (pH 8) and lyophilized. Five hundred yg dry weight of each fraction and 50 u g of each of 6 protein standards were used. The standards included cytochrome C, chymotrypsinogen A, ovalbumin, bovine serum albumin, aldolase, and catalase. Gels were cast in a 2 3 gel-casting tower in glass cassettes (8.2 X 0.27 X 8.2 cm) using either 7.5%, 10%, or 12% polyacrylamide. The acrylamide solution was mixed as described (Easterday e t al> 1976), poured into the casting tower and allowed to polymerize for 30 minutes. After polymerization the gel cassettes were stored in a humidity chamber at 4°C for 14-16 hours. Before electrophoresis all samples were treated with 20 pi of 0.01M tris with 1% SDS and 1% 2-mercaptoethanol (2-ME) and incubated for 15 minutes at 60°C. After incubation, 5 u l of a bromophenol blue4 sucrose solution was mixed with each sample; 10 samples were applied 1 TM Mol-Ranger , Pierce Chemical Co., Rockford, IL 2 Gel Slab Casting Apparatus GSC-8, Pharmacia Fine Chemicals, Piscataway. NJ 3 Gel Cassette Kit, Pharmacia Fine Chemicals, Piscataway, NJ 4 TM Bio-phore , Biorad Laboratories, Richmond, CA

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to each cassette. Electrophoresis was performed in a tris-glycine-SDS buffer at 90V in a specially designed chamber until the tracking dye moved to 1.5-2.0 cm from the bottom of the gel. When electrophoresis was complete the gels were removed from the glass cassettes, rinsed in distilled water and fixed in 25% isopropanol, 10% acetic acid for 48 hours.. After fixation, the gels were stained for 48 hours in 0.02% coomassie blue in 7% acetic acid; gels were destained with 25% isopropanol, 10% acetic acid until background color disappeared. The distance from the origin to the solvent front was measured as was the leading edge of each standard band and each distinct band of the individual fractions. The distance moved (mobility) of each standard of known molecular weight was plotted to obtain a standard curve for determining molecular weights of the fraction constituents. A standard curve for each polyacrylamide percentage was determined. Adjacent fractions from each of the MSSA, FSSA,and MFSSA with apparent identical constituents were combined. Polyacrylamide Gel Electrophoresis Fractions combined after PAGE-SDS were further examined using PAGE electrophoresis in 10% polyacrylamide with no SDS. Gels were cast as described above. Five hundred yg of each sample was solubilized in a tris-boric acid-EDTA electrophoresis buffer and 6 samples were applied to each cassette. Electrophoresis was performed at 120V until the solvent front was 1.5-2.0 cm from the bottom of the gel, then the gels were removed and split into halves with a wire cutter and gel-slicing Electrophoresis Apparatus GE-4, Pharmacia Fine Chemicals, Piscataway, NJ

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frame. Half of each gel was fix^d in 20« sulfosalicylic acid for 40 minutes then stained for protein constituents with coomassie blue as before. The other half of each gel was used to stain for presence of carbohydrates. Initially, a periodic acid Schiff's stain (PAS) was used (Maurer, 1971). These gels were fixed in a sodium periodate, glacial acetic acid, hydrochloric acid and TCA solution for 16 hours, then washed for 8 hours in a glacial acetic acid, TCA solution and 2 stained in Schiff s reagent for 16 hours. When the PAS stain was 3 complete, the same gel halves were stained with alcian blue in a 0.5":' solution in 3% acetic acid for 48 hours; destaining was with 3% acetic acid. A general scheme for antigen purification and characterization is illustrated in Figure 1. Humoral Immune Respons e Determinations Purification of the Indirect Hemagglutination Antigen Anti-P. invnitis antibodies were measured in all sera collected using a semipurified antigen prepared according to Sawada et at., (1965) and Mantovani and Kagan (1967). Adult D. immitis from naturallyand experimentally-infected dogs were separated by sex, washed 3 times in PBS, and extracted in 0.01M tris (pH 8) as described above. Equal protein quantities of each MSSA and FSSA were combined and 10% TCA was added until the pH reached 3.5. This mixture was left at 4°C Pharmacia Fine Chemicals, Piscataway, NJ 2 Fisher Scientific Co., Fair Lawn, NJ 3 Canalco, Inc., Rockville, MD

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Figure 1. Diagrammatic representation of the scheme used in purification and characterization of Dirofilaria irrmitii antigens.

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for 1 hour, then centrifuged at 12,100 X g for 45 minutes at 4°C; the supernatant was dialyzed against distilled water for 48 hours at 4°C. Approximately 120 mg of TCA soluble protein was lyophil ized and reconstituted to 20 ml with distilled water. Group separation on this preparation was at 4°C, in a glass chromatography column (2.5 X 1 2 60 cm) using Sephadex G-100 equilibrated with distilled water. Elution was with distilled water at a flow rate of 3 ml/hour. The 3 eluate was continuously monitored for protein at 254 nm and each 4 5 ml was collected using an automated fraction collector. Those fractions within the initial, principal protein peak were combined, lyophil ized, and resolubilized with 5 ml of 0.005M sodium acetate (pH 4.6). The sodium acetate solubilized preparation was layered onto a 5 carboxymethyl cellulose column equilibrated with sodium acetate (pH 4.6) and eluted at a flow rate of 20 ml/hour. The eluate was monitored and collected as above and after each protein fraction was completely eluted, the column was eluted with a buffer of increasing molarity or pH. The eluants used in succession were 0.005M sodium acetate (pH 4.6), 0.05M phosphate buffer (pH 6), 0.1M sodium chloride (pH 7), 0.2M sodium chloride (pH 7), 0.4M sodium chloride (pH 7), 0.1T hsC0 Chromatographic Column, ISC0, Lincoln, MB 2 Sephadex G-100, Pharmacia Fine Chemicals, Piscataway, NJ 3 UA-5 Absorbance Monitor, ISC0, Lincoln, NB 4 Model 328 Fraction Collector, ISC0, Lincoln, NB 5 CM-cellulose, Pharmacia Fine Chemicals, Piscataway, NJ

PAGE 36

sodium hydroxide (pH 7). The fractions eluted with each of these eluants were collected separately; previous to use in the indirect hemagglutination (IHA) assay the fractions eluted with 0.2M and 0.4M sodium chloride were combined and dialyzed against PBS (pH 7.2) for 48 hours at 4°C. The protein concentration of this preparation was determined previous to antigen titration. A scheme for the purification of the IHA antigen is illustrated in Figure 2. Indirect Hemagglutination Assay The IHA method was a modification of a widely used technique in the serodiagnosis of parasitic diseases (Kagan and Norman, 1976). Approximately 5 ml of sheep red blood cells (SRBC) suspended in Alsever's 1 2 solution were washed 3 times in hemagglutination buffer (HAB) (pH 7.3). 3 Centrifugation was for 10 minutes each time at 800 X g. After the final wash, the packed cells were adjusted to a 2.5% suspension in HAB 4 and mixed with an equal volume of 1:2 X 10 tannic acid solution. This mixture was incubated at 37°C for 10 minutes, then the cells were washed twice and resuspended to a 2.5% solution with HAB. The tanned cells were sensitized with one of five dilutions (1:4, 1:8, 1:16, 1:32, 1:64) of the column purified antigen by adding an equal volume of the antigen dilution to a 2.5% suspension of tanned SRBC; the mixture was incubated at 37°C for 15 minutes. The antigen-sensitized SRBC were washed twice and adjusted to a 1.5% suspension with HAB with 1% heat inactivated Becton, Dickinson and Co., Cockeysville, MD p Bacto Hemagglutination Buffer, Difco Laboratories, Detroit, MI 3 PR-2 Refrigerated Centrifuge, International Centrifuge Co., Needham Heights, MA

PAGE 37

Adult Di vofi lavi a inmi ti t Aqueous Soluble Somatic Extract Trichloroacetic Acid Treatment Discard Precipitate .005M Acetate Buffer pH 4.6 Supernatant Dialyzed vs. Distilled Water Gel Filtration-GlOO Cat ionExchange Chromatography on Carboxymethyl Cellulose .05M ' .1M ' .2M ".4M X .1M Phosphate Buffer NaCl NaCl NaCl NaOH pll 6 pH 7 pll 7. jjH 7 pll 7 Indirect Hemagglutination Antigen Figure 2. Diagrammatic representation of the procedure for purification of the Dirofilaria imrnitis indirect hemagglutination antigen

PAGE 38

fetal calf serum (FCS). Twenty-live jjI of the diluent, HAB with 2 1% FCS, was added to each well of a V-bottom microti ter plate in which serum dilutions were to be made. Fifty pi of either pooled canine immune serum or pooled canine serum from 4 dogs not infected (normal serum) were serially diluted with 25 y1 dilution loops. When the serum dilutions were complete, 25 )il of each antigensensitized SRBC suspension was added to each well in a dilution series of both normal and immune serum. The plates were incubated for 3 hours at 22°C; the highest serum dilution with hemagglutination was considered the titer. The antigen dilution giving the highest titer with the immune serum and no reaction with the normal serum was optimal; this dilution was used to determine anti-Z?. immitis antibody titers in individual serum samples collected over the course of the investigation. A diluent control was made by adding 25 pi of diluent and 25 pi of antigen-sensitized SRBC to 12 individual wells; a tanned SRBC control was made by adding 25 yl of unsensitized, tanned SRBC to each well of a dilution series of both normal and immune serum. In both controls it was necessary to obtain negative reactions. Differences in anti-£. irrmitis antibody titers of single infected, double infected, and noninfected dogs were statistically evaluated using a general linear models procedure (Steel and Torre, 1960). Grand Island Biological Co., Grand Island, MY 2 Cooke Laboratory Products, Alexandria, VA 3 Beckman Instruments, Inc., Palo Alto, CA

PAGE 39

Cel _l -Mediated Immune Response Determinations Lymp hocyte Isolation The lymphocyte isolation technique was a modification of the method of Thilsted and Shifrine (1977). Fifteen ml of blood was obtained by venipuncture from the cephalic vein of each dog and mixed with heparin (10 U/ml of blood). The anticoagulant treated blood was mixed with 15 ml of RPMI-1640; this mixture was divided in half, layered over 30 ml of a sucrose polymer-diatrizoate solution 2 with 0.1% methylcel lulose. The tubes were centrifuged at 800 X g for 30 minutes at 22°C. The resultant lymphocyte-rich layer was harvested with a sterile pipette and washed in 50 ml of RPMI-1640. Total leuckocyte counts were made using a hemacytometer, then 200 yl of the preparation was fixed on a microscope slide with a cyto-centrifuge, 3 stained 4 , and examined microscopically to obtain a differential leuckocyte count. Leukocytes were classified as either mononuclear or polymorphonuclear. After total mononuclear cell numbers were determined each cell preparation was washed in 50 ml of RPMI-1640 and resuspended to 3.0 X 1 6 mononuclear cells/ml in RPMI-1640 supplemented with 1% antibiotic5 antimycotic and 10% normal canine serum absorbed with human red blood cells (HRBC). This cell suspension was used for the lymphocyte-rosette assay, then adjusted to 1.25 X 10 mononuclear cells/ml for lymphocyte transformation. Grand Island Biological Co., Grand Island, NY 2 TM LSM Solution, Bionetics Laboratory Products, Kensington, 3 Shandon-Elliott, Sewickley, PA 4 Cameo Quick Stain, Scientific Products, McGraw Park, IL 5 Grand Island Biological Co., Grand Island, NY

PAGE 40

Lymphocyte Transformation Four major lymphocyte transformation experiments were performed 43, 45, 49, and 50 weeks after the initial D. imnitis infection. All experiments were initiated after patency of the first infection and after Group C was infected the second time. Two-tenths ml of each cell suspension was placed into each 1 5 predetermined well in a microculture dish to give 2.5 X 10 cells/well. Various levels of mitogen and antigen were assayed for lymphocyte transforming ability in quadruplicate cultures; unstimulated cultures from each cell preparation served as a background control. Phyto2 hemagglutinin P (PHA) was added in a range of 0.025-0.2 yl/culture, and Pokeweed Mitogen (PWM) and Concanavalin A (Con A) were added in ranges of 1-10 yl/culture and 1-10 yg/culture, respectively. The D. imnitis antigen used was a combination of MSSA and FSSA (1:1) in PBS (pH 7.2) and was used in a range of 10-100 yg of protein/culture. All mitogens and antigens were solubilized in RPMI-1 640 and were added to the respective cultures in a 10 yl volume. The cultures were 5 incubated for 48 hours at 37°C in 5% CO,-,, then 0.5 yCi of tritiated thymidine in RPMI-1 640 was added to each well and incubation was continued under the same conditions for 16-20 hours. When the incubation Cooke Laboratory Products, Alexandria, VA 2 Difco Laboratories, Detroit, MI 3 Grand Island Biological Co., Grand Island, NY 4 Miles Laboratories, Inc., Elkhardt, IN 5 Model C0-20, New Brunswick Scientific, New Brunswick, NJ The Radiochemical Centre, Amersham, England

PAGE 41

was complete, the cultures were collected individually on a paper strip using an automated culture harvester. The strip was air-dried and each portion of the paper with the cells from an individual culture was placed in a scintillation vial. The vials were filled with 4.5 ml of 2 a scintillation cocktail; after 3-6 hours of dark adaptation the radioactivity in each vial was measured using an automated liquid 3 scintillation counter. The amount of tritiated thymidine incorporated in cultures at each level of mitogen or antigen treatment was expressed as the mean counts per minute (cpm) of the 4 replications at each level. A stimulation index (mean cpm of stimulated culture/mean cpm of nonstimulated culture) was used to evaluate the extent of lymphocyte transformation. Preliminary evaluation of lymphocyte transformation data indicated that there was no difference between dogs receiving single or double D. immitis infections; all data on cell -mediated immune response determinations are expressed as noninfected dogs (Group A) and infected dogs (Groups B + C). Further, because of limited animal numbers and the variability in lymphocyte responses it was necessary to evaluate the data after all 4 lymphocyte transformation experiments were combined. Additionally, data on the lymphocyte responses to all levels of each antigen and mitogen were combined to evaluate differences in lymphocyte responsiveness. Differences in mitogen and antigen responses were analyzed using a general linear models procedure. ] 0tto Hi Her Co., Madison, WI 9 Fisher Scientific, Pittsburg, PA LS 330, Beckman Instruments, Inc., Palo Alto, CA

PAGE 42

Lymphocyte Rosette Assay A modification of the procedure of Bowles et at. (1975) was used to quantitate canine lymphocytes forming nonimmune erythrocyte rosettes with HRBC. This assay was performed with the same cell preparations used in the 4 lymphocyte transformation experiments. Two-tenths ml of a 0.5% suspension of HRBC in RPMI-1640 and a 0.2 ml aliquot of each lymphocyte suspension at 3.0 X 10 cells/ml were mixed in triplicate. This mixture was incubated at 22°C for 30 minutes then centrifuged at 200 X g for 5 minutes. The cell pellet was incubated at 4°C for 14 hours then gently resuspended; one drop of the suspension was mixed with one drop of a dilute acridine orange stain (Brostoff, 1974). The stained suspension was examined in a hemacytometer at 100X using fluorescent microscopy. One hundred fluorescing cells were counted and cells binding two or more erythrocytes were counted as rosetteforming cells. The percentage of rosetteforming cells in each cell suspension was expressed as the mean of the triplicate samples. I mmune Response to Sheep Red Blood Cells After the initial data on the lymphocyte transformation experiments were obtained, it was apparent that a comparison of the immune response of infected and noninfected animals to a heterologous antigen, SRBC, would be advantageous. Three ml of a 20'X SRBC suspension in PBS (pH 7.2) was administered intravenously to all dogs 49 weeks after the initial D. ivmitis infection and again 16 days later. Sera was collected on the day of the primary immunization and 8, 16, 20, 22, 27, and 29 days thereafter for anti-SRBC antibody determinations. Antibody was measured Eastman Kodak Co., Rochester, NY

PAGE 43

by direct hemagglutination. Two 25 pi aliquots of each serum sample were mixed with either 25 pi of HAB with 1% FCS or 25 pi of 0.1M 2-ME and incubated at 37°C for 1 hour (Scott and Gershon, 1970). After incubation, the sera was diluted in a microtiter system as described above and 25 pi of a 1.5% solution of SRBC was added to each well. Titers for untreated and 2-ME treated sera were determined after incubation at 22°C for 3 hours. This assay was performed twice on each serum sample and the mean titer was recorded. One hundred pi of sera from each dog 20 and 22 days after primary SRBC immunization was pooled and approximately 2 ml of this pool was separated by gel filtration on a Sepharacyl S-200 column (2.5 X 80 cm). The Sepharacyl S-200 was equilibrated and eluted with a sodium chloride, trizma base, hydrochloric acid buffer. The column flow rate was approximately 5 ml/hour and each 2.25 ml was collected 2 in individual tubes on an automatic fraction collector. Ultraviolet light absorbance at 280 nm of the eluate in each tube was determined and 4 pools corresponding to the manufacturer's predicted peaks for IgG, IgA, IgM and albumin fractions were collected. Each pool was concentrated with negative-pressure dialysis then assayed for 2-ME labile antibody as described above to insure that IgM was the only fraction demonstrating 2-ME lability. Differences in anti-SRBC antibody and 2-ME labile anti-SRBC antibody between infected and noninfected dogs were evaluated using a general linear models procedure. Pharmacia Fine Chemicals, Piscataway, NJ 2 Gilson Medical Electronics, Inc., Middleton, WI

PAGE 44

RESULTS Fecal examinations for helminth ova on all dogs were negative before and throughout the experimental period. All dogs remained healthy except for occasional minor lacerations obtained during fighting. All dogs in Groups B and C received 29-30 infective larvae each. Dogs in Group C were infected with an additional 30 larvae each at the second D. immitis infection. Microfilariae Counts Microfilariae were first detected 26 weeks after infection in one dog and were present in all infected dogs by 30 weeks after infection (Appendix I). One dog that received 2 D. immitis infections became amicrofilaremic 56 weeks after the initial infection and 22 weeks after the second infection; microfilariae were not detected in that dog again throughout the study (Appendix I). A marker dog was not used to detect the onset of patency of the second infection; there was no evidence of a difference in microfilariae counts between single and double infected dogs at any time (Figure 3). Antigen Purification and Characterization Pre parative Isoelectric Focusing The preparative, flatbed isoelectric focusing effected a good protein separation with a reproducible pH gradient and approximately a 70% protein recovery of the crude soluble somatic preparations. MSSA, FSSA, and MFSSA were initially divided into 17, 22, and 9 fractions, M

PAGE 45

3 Figure 3. Mean numbers (X10 ) of Dirofilca-ia immit-is microfilariae per ml of blood in Group B (single infection) and Group C (double infection).

PAGE 46

cr> cv rin m t y — oincoNmu-itrrooa ( t -oi xjaooia iw/aviyviiJoyoivN siimuu r=a on nvbw

PAGE 47

respectively, based on the isoelectric points and the staining intensity of apparent protein bands on the filter paper print. The filter paper print of each separation, the percentage of total recovered protein in each final fraction, and the pH gradient established during separation are illustrated in Figures 4-6. Immunodiffusion After immunodiffusion of the fractions separated by isoelectric focusing, the male, female, and microfilaria fractions were combined to 11, 14, and 6 fractions, respectively. If antigenic identity could be established, adjacent fractions were combined. Antigenic activity against both homologous rabbit antibody and canine antibody was present in most fractions (Tables 1-3). Adult worm fractions with the most apparent antigenic activity and complexity were within an isoelectric point (pi) range of approximately 4.8-6.5. Approximately 4 mg of TSF and 2 nig of TSM were recovered. There was some apparent antigenic activity when these were diffused against homologous rabbit antibody and canine antibody, but precipitin lines were weak and the degree of antigenic complexity could not be discerned. Polyacryl amide Gel Electrophoresis in Sodium Dodecyl Sulfate Adjacent fractions of each preparation with apparently identical protein constituents after PAGE-SDS were combined after consideration of isoelectric point ranges and antigenic identity. The male, female, and microfilaria fractions were combined to 7, 7, and 3 fractions, respectively, the final fraction number for each preparation. The approximate molecular weights of the protein constituents in the soluble somatic fractions are listed in Tables 1-3. The TSM and TSF preparations were less complex. The TSM had 2 protein constituents of

PAGE 48

Figure 4. Preparative isoelectric focusing separation and percentage of recovered protein in the final fractions from the Divofilaria irrmitis male soluble somatic extract.

PAGE 49

39 \ s. \ v Hd V \ -v \2\ ~5~ NI310Ud Q3d3A0D3d 30 39V±N30d3d

PAGE 50

Figure 5. Preparative isoelectric focusing separation and percentage of recovered protein in the final fractions from the Dirofilaria imdtis female soluble somatic extract.

PAGE 51

41 Hd \ V I 1 \ \ -r \ 17 \ 1 \ 2P \ NI310dd Q3d3AO03d 30 39VlN30d3d

PAGE 52

Figure 6. Preparative isoelectric focusing separation and percentage of recovered protein in the final fractions from the Dirofilavia immitis microfilaria soluble somatic extract.

PAGE 53

^ PERCENTAGE OF RECOVERED PROTEIN ro w -& en eo O O O O O PH

PAGE 54

o

PAGE 55

4-

PAGE 56

4-> +i m i CD

PAGE 57

4 molecular weight 7.7 and 5.2 X 10 ; the TSF had 5 protein constituents of 4 molecular weight 7.7, 7.1, 6.6, 5. 2, and 3.4 X 10 . Photographs of the separated fractions on polyacrylamide gel slabs appear in Appendix II. The standard lines for molecular weight determination obtained for each polyacrylamide gel percentage are in Appendices III-V. Polyacrylamide Gel Electrophoresis The number of proteins detected in individual fractions after PAGE are listed in Table 1-3. Two protein bands from the TSM preparation and 5 from TSF preparation were detected. Neither the PAS stain or the alcian blue stain were successful in detecting carbohydrate moieties associated with any of the separated protein constituents. The sample application point of all fraction and the electrophoresis tracks of the crude male and female preparations appeared to react with both stains, but staining was diffuse and could not be related to constituents of any of the fractions. Photographs of the PAGE separated soluble somatic fractions, crude soluble somatic preparations and Triton X-100 solubilized preparations are in Appendix VI Humoral Immune Response Thirty mg of the column-purified antigen preparation was obtained from the initial 120 mg of TCA-soluble preparation. A dilution of 90 ng of protein/ml was optimal in sensitizing SRBC with antigen. A titer of 1:512 with pooled canine immune sera and no titer with pooled canine normal sera were repeatedly obtained with SRBC sensitized with this antigen di lution. Anti-Z.). immitis antibody was first detected in Groups B and C 4 weeks after the initial D. immitis infection (Figure 7). Antibody

PAGE 58

Figure 7. Mean log ? of the reciprocal anti -Divofilca>ia immitis antibody titers in Groups A, B and C over the course of infection.

PAGE 59

83111 AQ08I1NV si(i> Q-llNf 1VD0HdO3H JO'tlOH NV3W

PAGE 60

levels in the infected groups were significantly higher (p<0.05) than in the uninfected group. The antibody titer in Group B, the single infection group, began to decrease shortly after patency (Week 30) and antibody levels in Group C, 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. Individual anti-D. immitis antibody titers are tabulated in Appendix VII. Cell -Mediated Immune Response Lym phoc y te Transform ation Peripheral blood lymphocyte transformation could not be induced in D. immitisinfected dogs with any level of D. immitis antigen tested (Table 4). A significantly (p<0.0001) depressed reactivity to PHA, PWM,and Con A was observed in lymphocyte cultures from infected dogs (Table 4). Lymphocyte Rosettes There were no differences evident in the mean percentage of rosette-forming cells between infected and noninfected dogs (Table 5). Although the standard deviation observed was low, and the mean percentage of rosettes remained reasonably consistent between experiments, the percentage of rosette-forming cells from the same dog often differed markedly between experiments. Imm une Response to Sheep Red Blood Cells There were no significant differences in anti-SRBC antibody levels between infected and noninfected dogs (Figure 8). Although there appeared to be a greater quantity of anti-SRBC 2-ME labile antibody after the secondary infection (Figure 9), it was not statistically

PAGE 61

TABLE 4. Mean counts per minute and stimulation indices from four canine peripheral blood lymphocyte transformation experiments of Dirofilai'ia immitit infected dogs and noninfected dogs. Cul ture Treatment Group A noninfected dogs) Groups B + C infected dogs^ none .025]jl PHA . 05ul PHA .10yl PHA .20yl PHA C lyl PWM 5yl PWM 10m 1 PWM lpg Con A 5pg Con A lOyg Con A 10ug DIA e 50ug DIA lOOng DIA 84 90 2152

PAGE 62

TABLE 5. Percentage of canine peripheral lymphocytes forming nonimmune rosettes with human erythrocytes. Experiment Group A [noninfected dogs' Groups B + C [infected dogs 24.7 + 5.5 18.2 + 1.8 26.7 + 5.2 19.9 + 5.5 24.6 + 5.7 33.2 + 5.8 31.6 + 4.8 28.8 + 5.8 Data is expressed as the mean leukocyte percentage of rosetteforming cells + the standard deviation of triplicate samples from each doq.

PAGE 63

Figure Mean log„ of the anti-sheep red blood cell antibody titers in Group A and Groups B + C.

PAGE 64

H31I1 AQ09I1NV 3'8'yS "I1NV 1V30ddlD3d do'ocn NV3W

PAGE 65

Figure 9. Mean difference of log ? of reciprocal anti-sheep red blood cell antibody titers before and after serum treatment with 2-mercaptoethanol .

PAGE 66

a

PAGE 67

significant. The average anti-SRBC antibody titers before and after 2-ME treatment for each dog are listed in Appendix VII. A good separation of the pooled anti-SRBC on Sepharacyl S-200 was effected (Figure 10). Anti-SRBC antibody activity was detected in Pools A, B,and C. Pool A and Pool C demonstrated the predominance of antiSRBC activity. After 2-ME treatment the mean anti-SRBC antibody titer decreased from 1:8 to 1:1.5 in Pool A, the IgM pool. Antibody titers in Pool B, the IgA pool, and Pool C, the IgG pool, remained unchanged after 2-ME treatment.

PAGE 68

Figure 10. Separation profile of pooled canine anti-sheep red blood cell serum on Sepharacyl S-200 and the pools collected for 2-mercaptoethanol lability determinations.

PAGE 70

DISCUSSION Antigen Purification and Characterization Any definitive conclusions on the antigenic relationships of different D, immitis life-stages are not possible when the data from this investigation are evaluated; however, this study contributed to progress toward that end. The degree of antigenic complexity detected in the different life-stages is remarkable and with the partialcharacterization data it will be helpful when further studies on antigenic relationships are considered. Separating the myriad of proteins present in each crude soluble somatic extract with preparative isoelectric focusing proved to be advantageous. Recent reports have described disc electrophoresis, immunoelectrophoresis and countercurrent immunoelectrophoresis techniques that were successful in delineating the protein, and antigenic complexities of crude D. immitis extracts (Wheeling and Hutchison, 1971; Desowitz and Una, 1976). The principal deficit in the findings in both reports was that the separated and partially characterized fractions could not be directly related to immunological activity. In the present study, the fractions recovered after isoelectric focusing of large quantities of protein were easily harvested and could be used individually in immunological and biochemical characterization techniques. There were antigens in all of the adult fractions and 2 of the 3 microfilaria fractions. The antigens were demonstrable using either

PAGE 71

homologous rabbit antisera or canine immune sera; the predominance of antigenic activity and complexity in the adult worm fractions was in the pi range of approximately 4.8-6.5. In any further studies, the ampholines used in preparative isoelectric focusing could be confined to this pH range to concentrate on a more complete separation in this area. Since Immunoelectrophoresis demonstrated at least 10 precipitin arcs in the crude adult D. immitis preparation tested against homologous rabbit antisera (Wheeling and Hutchison, 1971), further separation of the soluble somatic fractions will be necessary before antigenically distinct fractions are obtained. PAGE-SDS was effective in determining approximate molecular weights as well as comparing the protein-staining constituents of adjacent fractions. Although there was some contamination evident in the proteins used in establishing the standard lines for molecular weight determinations, reliable lines could be established. The lowest molecular weight standard, cytochrome C, traveled into the solvent front in the 7.5% polyacryl amide resulting in a point inconsistent with the standard line (Appendix III). The 2 lowest molecular weight standards did not fit the standard line well in the 12% gels (Appendix V), but the line was consistent in the molecular weight range of most proteins present in the fractions. It is likely that the 12% polyacrylamide had too much cross-linking bis-acrylamide resulting in this effect (Weber and Osborn, 1969). Stains for carbohydrate after PAGE were unsuccessful. Some diffuse staining with PAS and alcian blue was noted in the crude fractions; the staining appeared to be dispersed over the entire electrophoresis track

PAGE 72

in each case. Additionally, there was some staining evident at the application point of each fraction. Wheeling and Hutchison (1971) reported 2 bands of carbohydrate-staining activity after disc electrophoresis of a crude adult extract; one band was near the origin and one was near the solvent front. It is possible that disc electrophoresis yielded sharper, more dense bands than the PAGE resulting in a more confined, intense stain. It was unusual to note that in fraction F-l and MF-1 more protein constituents were detected after PAGE than after PAGE-SDS. The 2-ME reduction before PAGE-SDS may have reduced large macromolecules to a number of small molecular weight proteins that traveled with the solvent front or different macromolecules may have had constituents of similar molecular weight that moved a common distance after reduction and PAGE-SDS. The Triton X-100 solubilization of adult worm cuticles was largely unsuccessful. Unfortunately, only limited quantities of protein could be recovered. These preparations contained few proteins; relatively only a few bands appeared on PAGE and PAGE-SDS gels. Both preparations appeared to be weakly antigenic against both homologous rabbit antisera and canine immune sera when compared to soluble somatic preparations. Some success with Triton X-100 solubilization has been reported with Onchocerca volvulus antigens (Marcoullis and Grasbeck, 1976), but it was not clear if the worm preparations were thoroughly extracted in an aqueous system before Triton X-100 solubilization. It is possible that some of the antigenic complexity reported was due to residual aqueous soluble proteins. Future work on the characterization of these soluble somatic antigen fractions should make use of immunologic techniques. After

PAGE 73

having gained some information on the protein makeup, comparisons of the antigenic components of the fractions are necessary to learn about the possible stageor sex-specific antigens. Antigenic comparisons of each fraction with every other fraction could be considered, but initially it would be important to compare those fractions from different preparations within the same isoelectric point range. Comparative immunodiffusion would be a logical technique to use in making the comparisons, but interpretation may be difficult. A radioimmunoassay technique has been successfully used in the determination of stage and species specificity of Schistosoma mansoni antigens (Hamburger et at., 1976). This technique would be ideal for antigenic comparisons of the D. imnitis fractions, but a greater degree of purity in the fractions will be necessary before the technique could be implemented. Hu moral Immune Respon se Results obtained on antibody titers were largely comparable to data from other similar studies (Pacheco, 1966; Weiner and Bradley, 1972). Anti-Z). immitis antibody was detectable between 2 and 4 weeks after infection and peak titers were reached 2 weeks after D. imnitis reached patency in all dogs. It would be difficult to interpret, as Pacheco (1966) did, that microfilariae may be responsible for absorbing antibody and removing it from peripheral circulation, but the onset of tiie microfilaremia is obviously associated with the decrease in circulating antibody. Since the titers diminish 2-4 weeks after the appearance of microfilariae, the microfilariae may be directly affecting the immune system and the decline of antibody titers could be due to

PAGE 74

the normal, biological half-life of the specific immunoglobulins. Weiner and Bradley (1972) reported that a second D. immitis infection did not result in a typical anamnestic response, although dogs infected a second time had prolonged antibody levels when compared to dogs with single infections. The results illustrated in Figure 7 indicated that there was a delayed anamnestic response in the group receiving 2 infections, but that response was not statistically significant (p>0.05). On close observation, this apparent secondary response was related to unusually high antibody titers in the dog that became amicrofilaremic (Appendix VII). This animal developed high antibody titers at Week 50, just prior to the amicrofilaranic state and at the same point of the apparent anamnestic response. The unusual antibody response in this animal accounts for the artifact (Figure 7) when the antibody levels are illustrated; it also contributes to the enigma of the relationship of the microfilariae to the canine immune response during the progress of infection. Mantovani and Kagan (1967) reported that the TCA soluble antigen used in the present investigation was both genusanc' species-specific based on IHA and skin testing of naturally-infected dogs and Pacheco (1966) reported that an acid-soluble preparation of saline extracted adult D. inmibis was highly specific in serologic testing of experimentallyinfected dogs. It is impossible to assess the specificity of the purified, acid-soluble antigen preparation used without testing it against sera from dogs or other animals infected with other parasites, but evidence indicates that it is more specific than the crude preparations typically

PAGE 75

used. The I HA titers for non infer; ted dogs reported by Weiner and Bradley (1972) were often as high as 1:256 with a crude saline-soluble antigen, making interpretations of antibody changes very difficult. On this basis alone, the purified antigen used in the present study was more specific; anti-D. irmnitis antibody titers of the noninfected animals never exceeded 1:2. Further, the antigen preparation 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 and Bradley, 1972). Based on these results, future work on the humoral immune response to D. irmnitis should be concentrated on a more specific and well-defined antigen and a better serologic test. As research progresses on the isolation and characterization of D. immitis antigens a stage-specific antigen may be obtained. Detection and quantitation of adult and microfilaria-specif ic antibodies over the course of infection may answer questions on the decrease in antibody after the appearance of microfilaria and on the nature of the antibody response preceeding the amicrofilaremic state in some dogs. The enzyme-linked immunoabsorbent assay has been used in serologic testing of Onchocerca volvulus (Bartlett et al., 1975), another filarid parasite; if it is as sensitive and reproducible as reported for other systems (Voller et al., 1976), it may be ideal for antibody determinations in D. immitis infections. Cell-Mediated Immune Response No evidence of D. immitis antigen-induced lymphocyte transformation of peripheral blood lynphocytes of infected dogs was obtained. In

PAGE 76

addition to the antigens used in the 4 major lymphocyte transformation experiments, the acid-soluble IHA antigen and a crude 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 et al., 1977; Portaro et al., 1977). Ottesen et al. (1977) used antigens from saline extracted D. immitis and Brugia malayi adults to successfully transform peripteral blood lymphocytes from humans infected with Wuohereria bancvofti. Mean stimulation indices as high as 36 were recorded when D. immitis antigens were used to transform peripheral blood lymphocytes of noninfected, W. bancvoftiexposed patients. Portaro et al. (1977) have used saline extracts of D. immitis j Tvichnella spiralis and Brugia pahangi to transform splenocytes from B. pahangi infected jirds. Each of the filarial antigen preparations induced lymphocyte transformation but the T. spiralis antigen did not. Of the 2 filarial antigens, the B. pahangi preparation was more effective in transformation. These data suggested a possible f ilaria-specific diagnostic test and confirmed that specific antigen-induced lymphocyte transformation was possible in a filarial system. The failure to induce D. immitis -specif ic lymphocyte transformation in this study may have been related to a highly significant (p<0. 0001 ) depression of mitogen responsiveness in infected dogs. Depressed mitogen responses have been reported in such diverse disease situations as cancers (Mannick et al., 1977), leishmaniasis (Farah et al., 1976), and malaria (Spira et al., 1976). Several nonfilarial helminths have been associated with an immune supression phenomenon including T. spiralis

PAGE 77

(Cypess et al., 1973; Faubert and Tanner, 1975), Nematospiroides dubius (Shimp et al., 1975), Asaaris suwn (Crandall and Crandall, 1976), Taenia cvassiceps (Good and Miller, 1976), and Schistosoma mansoni (Pelley et at, 1976). Two diseases of dogs, demodectic mange (Scott, et at, 1974 and 1976) and canine distemper (Krakowka et al,, 1975) have been associated with diminished mitogen responsiveness. Scott et al. (1974) reported a correlation between low mitogen responsiveness and Demodex canis infection and later work (Scott et al., 1976) indicated that successful treatment for removal of the mite would reverse the phenomenon. Recently, various types of immune suppression have been documented for 3 other filarid parasites (Dalesandro and Klei, 1976; Ottesen et al., 1977; Portaro et al., 1976). Dalesandro and Klei (1976) using Dipetalonema vitae 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. Ottesen et al. (1977) claimed that a specific cellular unresponsiveness to W. hanevofti occurred in infected humans. Humans with infections, humans without infections but exposed to the parasite, and humans with no exposure to the parasite were immunized with tuberculin (PPD) and streptococcal (SK-SD) antigens. Lymphocyte responses to those antigens, filarial antigens and mitogens revealed that only the response to filarial antigens was impaired in infected humans; mitogen, PPD,and SK-SD responses were all normal. In other work, splenocytes from B. pahangi-i nfected jirds were shown to transform with exposure to filarial antigens, but mitogen responsiveness was

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inversely correlated to the appearance of f ilarial-specif ic splenocytes (Portaro et at. , 1976). It is possible that D. immitis antigen-induced lymphocyte transformation may have been observed if such experiments were attempted before patency; evidence (Dalesandro and Klei, 1976) would indicate that the immune suppression phenomenon may be associated with the microfilaremia. The decline in anti-O. immitis titers shortly after the onset of the microfilaremia would support the idea of a microti laria-associated immune suppression. Further work should concentrate on the role of the microfilariae in this effect. Since the 3 mitogens used in this study were either T-cell mitogens or T-cell dependent B-cell (thymus-independent lymphocyte) mitogens in other organisms, an impaired T-cell function or a decreased number of T-cells in the infected animals would explain the reduced responses. Enumeration of canine T-cells by nonimmune rosette formation with HRBC was reported (Bowles et at., 1975) and this technique was implemented in this investigation. There were no differences in the percentage of rosette-forming cells between infected and noninfected dogs. Recent work (Krakowka and Guyot, 1977) demonstrated that canine eosinophiles formed nonimmune rosettes with HRBC. In the rosette procedure used in this investigation, acridine orange stain was used for ease of counting the cells. At a magnification of 100X leukocyte nuclear morphology was not easily determined thus a small percentage of the cells counted as lymphoid rosettes may have actually been eosinophilic leukocytes. Experiments were performed to examine the rosettes at high magnification and the results confirmed the work of Krakowka and Guyot (1977). The slightly

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higher percentage of rosetteform ing cells in infected dogs was probably due to more contaminating eosinophiles in the mononuclear cell preparation because of the chronic eosinophilia (Weiner and Bradley, 1972) in D. irnmitisinfected dogs. In an attempt to assay T-cell function in D. irnmitis -infected dogs a heterologous T-cell dependent antigen, SRBC, was used to immunize the dogs and measure potential differences in antibody responses to that antigen. There were no differences between the groups in total hemagglutinating antibody or 2-ME labile hemagglutinating antibody. The 2-ME labile antibody responses seemed to occur primarily in the IgM class of immunoglobulin after secondary immunization of infected dogs and in the IgG class in noninfected dogs. This observation would support the hypothesis of impaired T-cell function, but it could not be substantiated with statistical analysis. Unfortunately, it was not possible to do antigen dose response studies of SRBC in the canine before immunization and the dose may have been too high; T-cell dependence or independence of the immune response to this antigen has been shown to be antigen concentration-dependent (Playfair and Purves, 1971; Lemmel et at., 1971). The implications of a state of diminished immune responsiveness in D. irnmitisinfected dogs are numerous. This phenomenon must be considered in evaluating the pathogenesis of D. irnmitis infections. It is reasonable to assume that this condition will predispose an infected dog to infectious and neoplastic diseases. Further, even though preliminary experiments on immunizing dogs with irradiated larvae have shown promise (Wong el a'/., 1974), the results of the present investigation demonstrate that the complexity of the immune response to

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D. immitis should be more clearly defined before it can be effectively manipulated to prevent infections. One of the most interesting considerations in view of the -depressed immune response is the possibility of treating the infection with an agent that will potentiate the immune response in conjunction with, or beyond, killing the parasite. Levamisole, a compound often used in immunologic enhancement (Bruley-Rosset, 1976), has been highly effective in killing D. immitis microfilariae (Bradley, 1976) and variably effective against adult worms (Boring and Shepard, 1974). The mode of action of levamisole has not been ascertained, but may be related to an enhanced immune responsiveness. This principle has been investigated in the treatment of human filariases (Pinon et al., 1974), but inconclusive results were obtained. Experiments evaluating the effectiveness of an immune-enhancing agent against D. immitis in dogs may provide information to aid the treatment of human filariases as well as canine dirofilariasis.

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CONCLUSIONS Soluble somatic extracts of D. immitis males, females, and microfilariae were separated into 7, 7, and 3 fractions, respectively. The isoelectric point range, the number of protein constituents after PAGE and PAGE-SDS and the estimated molecular weight of each constituent after PAGE-SDS were determined. A good deal of complexity was present in most fractions as evidenced by specific protein staining of the polyacrylamide gels; stains to detect carbohydrate moieties were unsuccessful. Antigenic activity was demonstrated in each adult D. immitis fraction and 2 of 3 microfilarial fractions. Triton X-100 solubilization of adult D. immitis cuticles yielded preparations that were weakly antigenic with few protein constituents. A TCA-soluble column-purified antigen was demonstrated to be more effective in the IHA test than previously used antigens. Antibody titers in experimentally-infected dogs decreased after the appearance of microfilariae. Titers from dogs infected only once dropped and remained at low levels following the appearance of microfilariae, but antibody levels in dogs infected a second time were demonstrable throughout the study. D. immitis antigen induced lymphocyte transformation could not be effected in experimentally-infected dogs. This finding may have been related to a significantly (p<0. 0001 ) depressed mitogen reactivity of peripheral blood lymphocytes of D. imnitisinfected dogs. Quantitation of nonimmune T-cell rosettes and immunization of dogs with SRBC to assay 71

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T-cell numbers and T-cell function, respectively, did not reveal any differences in infected and noninfected dogs.

PAGE 83

APPENDICES

PAGE 84

4-1 U rOJ C <4OO r—

PAGE 85

i — CVI

PAGE 86

Appendix II. Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate: Crude Antigen Preparations and Soluble Somatic Fractions A. 7.5% polyacryl amide Cytochrome C Chymotrypsinogen A Ovalbumin Bovine serum albumin Aldolase Catalase Crude male Crude female C. 12% polyacryl amide 1

PAGE 87

77 12 3 4 5 6 7 8 1 23456789 9m 4 1 23456789 10 1 23456789 10 I • 12 3456789 10 1 2 3 4 5 6 7 8

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Appendix III. Standard Molecular Weight Curve for 7.5% Polyacrylamide Gel A= Bovine Serum Albumin B= Catalase C= Ovalbumin D= Aldolase E= Chymotrypsinogen A F= Cytochrome C 78

PAGE 89

UJ < Cl. o CO o to 1 — I ' I ' I ' I ' I Off) co h to ^ ~ I r rO (^01 x)iH9GM avinoaiow

PAGE 90

Appendix IV. Standard Molecular Weight Curve for 10% Polyacryl amide Gel A=

PAGE 91

o Lo o "rO O 'CVI I ' I ' I ' I Off) CO N -j— i [ m (^01 x ) 1H9GM dvino3now

PAGE 92

Appendix V. Standard Molecular Weight Curve for 12% Polyacrylamide Gel A= Bovine serum albumin B= Catalase C= Ovalbumin D= Aldolase E= Chymotrypsinogen A F= Cytochrome C o?

PAGE 93

I ' I ' I ' I ' I ' I ' — i — ' — r O CD 00 NtD ID «t rO -1 r O o

PAGE 94

Appendix VI. Polyacryl amide Gel Electrophoresis: Crude Antigen Preparations and Soluble Somatic Fractions A. 1 . Crude male 2. Crude female 3. Crude microfilaria 4. Male 1 5. Male 2 6. Male 3 B. 1. Male 4 2. Male 5 3. Male 6 4. Male 7 5. Female 1 6. Female 2 C. 1 . Female 3 2. Female 4 3. Female 5 4. Female 6 5. Female 7 6. Microfilaria 1 D. 1 . Microfilaria 2 2. Microfilaria 3 3. Triton X-100 male 4. Triton X-100 female 84

PAGE 95

85 123456 123456 • 'i 1 2 3 4 S 6 12 3 4 — . — ^f

PAGE 96

oocmcm^tcmcmcm ' — COi — *d" CO CO CVI i — , — CO =3" o o =3cvi cm cvi . — *dcvi cvi . — cm O-vfCMCO^CMCMr— CM CO CM O cvj , — co =d^f cvi <^tI «3csj < — — i— o^t-oo^-^tO C\J CVI CVI O O * CM CO ^J" "d" I — CVI CM CO UD n cm un i— o o cvi o CVJ O O CVJ CVJ O r— O O CVI r— CVI O CVJ O O CVI O i — CVI CM CM CM r— CVI O r—

PAGE 97

rC\J CVJ OJ Ovl «3" ^t" CVJ CVJ r-NWNIMN!j^^ W NNW 1 i — C"> Cvl CVJ O r— NN^^-^^^N^wpJNlfl^ CViCVICVJCVJCVI'^-CO'^CVICvJCVICVlCVIO ^" UD Cvl Cvl <3^1COCO^J-UD^t-OOCVJCVJCVJ^tcvi
PAGE 98

Q CO co c\j cvi c\j ^a«^f Cvl i — CNJ cvi c\j
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00 £= 3 >>-'E ro SE Q Q. t— i +J

PAGE 100

APPENDIX IX Abbreviations Defined and Used in the Text. B-cell Thymus independent lymphocyte Con A Concanaval in A cpni counts per minute DIA Dirofilavia imnitis antigen EDTA Ethylenediaminetetracetic acid FCS Fetal calf serum FSSA Female soluble somatic antigen HAB Hemagglutination buffer HBSS Hank's balanced salt solution HRBC Human red blood cells IgG Immunoglobulin class G IgM Immunoglobulin class M IHA Indirect hemagglutination MFSSA Microfilaria soluble somatic antigen MSSA Male soluble somatic antigen PAGE Polyacrylamide gel electrophoresis PAGE-SDS Polyacrylamide gel electrophoresis in sodium dodecyl sulfate PAS Periodic acid Schiff's stain PBS Phosphate buffered saline PHA Phytohemagglutinin P PPD Purified protein derivative-tuberculin antigen PWM Pokeweed mitogen

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Abbreviations (Continued) SDS Sodium dodecyl sulfate SK-SD Streptokinase-streptodornase antigen SRBC Sheep red blood cells T-cell Thymus-dependent lymphocyte TCA Trichloroacetic acid tris 2-amino-2(hydroxymethyl )-l ,3-propanediol TSF Triton X-100 solublized fractions of female cuticles TSM Triton X-100 solubilized fractions of male cuticles 2-ME 2-mercaptoethanol

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BIBLIOGRAPHY Adcock, J.L. 1961. Pulmonary arterial lesions in canine dirofilariasis. Am. J. Vet. Res. 22:655-662. Anonymous. 1968. Procedures, techniques and apparatus for electrophoresis. Gelman Instrument Co., Ann Arbor, MI Bartlett, A., D.E. Bidwell, and A. Voller. 1975. Preliminary studies on the application of enzyme immunoassay in the detection of antibodies in onchocercosis. Tropenmed. Parasit. 26:370-374. Boring, J.G. and E.H. Shepard, Jr. 1974. Effectiveness of levamisole hydrochloride as an adulticidal drug for Dirofilaria immitis . p. 89-92. In, Heartworm Symposium, 1974. Veterinary Medicine Pub. Co., Bonner Springs, KS Bowles, C.A., G.S. White, and D. Lucas. 1975. Rosette formation by canine peripheral blood lymphocytes. J. Immunol. 114:399-402. Bradley, R.E. 1976. Levamisole resinate as a Dirofilaria immitis microf ilaricide in dogs. J. Am. Vet. Med. Assoc. 169:311-316. Brostoff, J. 1974. A simple technique for counting rosettes using acridine orange. J. Immunol. Meth. 5:303. Bruley-Rosset, M., I. Florentin, and G. Mathe. 1976. In vivo and in vitro macrophage activation by systematic adjuvants. Agent Act. 6:251. Carlisle, C.H., C.W. Prescott, P.J. McCosker, and A. A. Seawright. 1974. The toxic effects of thiacetarsamide sodium in normal dogs and in dogs treated with Dirofilaria immitis. Aust. Vet. J. 50:204-208. Casey, H.W., O.K. Obeck, and G.A. Splitter. 1972. Immunopathology studies on canine heartworm disease, p. 31-32. I_n, R.E. Bradley and G. Pacheco (eds.), Canine Heartworm Disease: The Current Knowledge. University of Florida, Gainesville, Florida. Crandall, C.A. and R.B. Crandall. 1976. Ascaris suum: Immunosuppression in mice during acute infection. Exp. Parasit. 40:363-372. Cypess, R.H., A.S. Lubiniecki, and W. flcD. Hammon. 1973. Immunosuppression and increased susceptibility to Japenese B encephalitis virus in Tin chin el la spiralis-^ nfected mice. Proc. Soc. Exp. Biol. Med. 143:469-473.

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Dalesandro, D.A. and T.R. Klei. 1976. Evidence for immunodepression of Syrian hamsters and mongolian jirds by Dipetalonema viteae infections. Trans. Roy. Soc. Trop. Med. Hyg. 70:534-535. Desowitz, R.S. and S.R. Una. 1976. The detection of antibodies in human and animal filariases by countercurrent Immunoelectrophoresis with Dirofilaria immitis antigens. J. Helminth. 50:53-57. Donahue, J.M.R. 1975. Experimental infection of cats with Dirofilaria immitis. J. Parasit. 61:599-605. Easterday, R.L., M. Naphas, and I.M. Easterday. 1976. Manufacturer's Communication. Pharmacia Fine Chemicals, Inc., Piscataway, NJ Ellsworth, J.H. and A.H. Johnson. 1973. Precipitin test and soluble antigen fluorescent antibody technique for detecting Dirofilaria immitis in dogs. Am. J. Vet. Res. 24:689-692. Farah, F.S., S. Lazary, and A. DeWeck. 1976. The effect of leishmanial tropica on stimulation of lymphocytes with phytohaemaggluti ni n. Immunol. 30:485-489. Faubert, G. and C.E. Tanner. 1975. Leucoaggl utination and cytotoxicity of the serum of infected mice and of extracts of T. spiralis larvae and the capacity of infected mouse serum to prolong skin allografts. Immunol. 20:1041-1050. Foil, L. and T.C. Orihel. 1975. Dirofilaria immitis (Leidy, 1956) in the beaver, Castor canadensis. J. Parasit. 61:433. Forrester, D.J., R.F. Jackson, J.F. Miller, and B.C. Townsend. 1973. Heartworms in captive California sea lions. J. Am. Vet. Med. Assoc. 163:568-570. Franks, M.B. and N.R. Stoll . 1945. The isolation of microfilariae from blood for use as antigen. J. Parasit. 31:158-162. Good, A.H. and K.L. Miller. 1976. Depression of the immune response to sheep erythrocytes in mice infected with Taenia crassiceps larvae. Infect. Immun. 14:449-456. Hamburger, J., R.P. Pel ley, and K.S. Warren. 1976. V, 'chi stoma mans oni soluble egg antigens: Determination of the stage and species specificity of their serologic reactivity by radioimmunoassay. J. Immunol. 117:1561-1566. Harboe, N. and A. Ingild. 1973. Immunization, isolation of immunoglobulins, estimation of antibody titer, p. 161-164. In, A Manual of Quantitative Immunoelectrophoresis: Methods and Applications N.H. Axelsen, J. Kroll, B. Weeke (eds.) Universi tetsforlaget, Oslo, Norway.

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Harris, T.N., S. Harris, and C.A. Ogburn. 1971. Solubilization of H-2 histocompatibility antigens of the mouse by triton X-100 and butanol. Transplant. 12:448-458. Jackson, R.F. 1969. Diagnosis of heartworm disease by examination of the blood. J. Am. Vet. Med. Assoc. 154:374-376. Johnson, C.A. 1975. Ursus amevicanus (black bear) a new host for Divo filar ia itnmitis . J. Para sit. 61:940. Kagan, I.G. 1963. A review of immunologic methods for the diagnosis of filariasis. J. Parasit. 49:773-778. Kagan, I.G. and L. Norman. 1976. Serodiagnosis of parasitic diseases. Manual of Clinical Immunology. Am. Soc. Micro. Chap. 51:382-409. Klei, T.R., W.A. Crowell , and P.E. Thompson. 1974. Ul trastructural glomerular changes associated with filariasis. Am. J. Trop. Med. Hyg. 23:608-618 Klein, J . B . and E . D . S todda rd . 1977. Dirofi laria irnmi tis recovered from a horse. J. Am. Vet. Med. Assoc. 171:354-355. Kobayakawa, T. 1975. Cell-mediated immunity to Dirofilaria itnmitis . Japan. J. Med. Sci. Biol. 28:11-22. Krakowka, S., G. Cockerell, and A. Koestner. 1975. Effects of canine distemper virus infection on lymphoid function in vitro and in vivo. Infect. Immun. 11:1069-1078. Krakowka, S. and D.J. Guyot. 1977. Rosette formation assays in dogs: Lack of specificity of E rosettes for T lymphocytes. Infect. Immun. 17:73-77. Kume, S. 1974. Experimental observations on seasonal periodicity of microfilariae, p. 26-31. In, Heartworm Symposium, 1974. Veterinary Medicine Pub. Co., Bonner Springs, KS Lemmel, E.M., M.D. Cooper, and R.A. Good. 1971. Neonatal thymectomy and the antibody response to sheep erythrocytes in mice. Int. Arch. Allergy 41:873-882. Levine, N.D. 1968. Nematode parasites of domestic animals and of man. Burgess Pub. Co., Minneapolis, MM p. 451-454. Lindsey, J.R. 1965. Identification of cani ne microti lariae. J. Am. Vet. Med. Assoc. 146:1106-1114. Mannick, J. A., M. Constantian, D. Pardrige, I. Saporoscheta , and A. Badger. 1977. Improvement of phytohemaggl utini n responsiveness of lymphocytes from cancer patients after washing in vitro. Cancer Res. 37:3066-3070.

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Mantovani, A. and I.G. Kagan. 1967. Fractionated Dirofitaria immitis antigens for the differential diagnosis of canine filariasis. Am. J. Vet. Res. 28:213-217. Marcoullis, G. and R. Grasbeck. 1976. Preliminary identification and characterization of antigen extracts of Onchocerca volvulus. Tropemned. Parasit. 27:314-322. Masuya, T. 1976. Studies on the mechanism of the filarial periodicity: The autofluorescence in the microfilariae and their periodicity. Japan. J. Parasit. 25:283-312. Maurer, H.R. 1971. Disc electrophoresis and related techniques of polyacrylamide gel electrophoresis. Walter de Gruyter, New York, NY Medway, W. and T.C. Wieland. 1975. Dirofilaria immitis infection in a harbour seal. J. Am. Vet. Med. Assoc. 167:549-550. Neppert, J. 1974. Cross-reacting antigens among some filariae and other nematodes. Tropenmed. Parasit. 25:454-463. Orihel, T.C. 1961. Morphology of the larval stages of Dirofilaria immitis in the dog. J. Parasit. 47:251-262. Ottesen, E.A., P.F. Weller, and L. Heck. 1977. Specific cellular immune unresponsiveness in human filariasis. Immunol. 33:413-421. Otto, G.F. 1974a. Changing georgraphic distribution of heartworm disease in U.S. p. 1-2. Yn, Heartworm Symposium, 1974. Veterinary Medicine Pub. Co., Bonner Springs, KS Otto, G.F. 1974b. Occurrence of the heartworm in unusual locations and in unusual hosts, p. 6-13. In, Heartworm Symposium, 1974. Veterinary Medicine Pub. Co., Bonner Springs, KS Pacheco, G. 1966. Progressive changes in certain serological responses to Dirofilaria immitis infection in the dog. J. Parasit. 52:311-317. Pacheco, G. 1974. Relationships between the number of circulating microfilariae and the total population of microfilariae in a host. J. Parasit. 60:814-818. Pelley, R.P., J.J. Ruffier, and K. Warren. 1976. Suppressive effect of a chronic helminth infection, schistosomiasis mansoni, on the in vitro responses of spleen and lymph node cells to the T-cell mitogens phytohemagglutini n and concanavalin A. Infec. Immun. 13:1176-1183. Pinon, J.M.. M. Danis, and M. Gentilini. 1974. Trials of inimunostimulants in human filariasis. Proc. Third Interntl. Cong. Parasit. 2:1049.

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Playfair, J.H.L. and E.C. Purves. 1971. Antibody formation by bone marrow cells in irradiated mice. I. Thymus-dependent and thymus-independent responses to sheep erythrocytes. Immunol. 21:113-121. Portaro, J.K., S. Britton, and L.R. Ash. 1976. Brugia pahangi: Depressed mitogen reactivity in filarial infections in the jird, Meriones ungul aula bus . Exp. Parasit. 40:438-446. Portaro, J.K., S. Britton, and L.R. Ash. 1977. Use of lymphocyte transformation for the detection of filarial infections. J. Parasit. 63:173-174. Quails, D.R., P.J. Neuhaus, J.R. Athey, and M.J. Sweeney. 1975. A rapid batch method for the production of specific fluorescein isothiocyanate-labeled globulins to Dirofilaria imnitis microfilariae. Am. J. Vet. Res. 26:235-236. Sawada, T., K. Takei, D. Katamine, and T. Yoshimura. 1965. Immunological studies on filariasis. III. Isolation and purification of antigen for intradermal skin test. Japan. J. Exp. Med. 35:125-132. Sawada, R., K. Sato, and S. Sato. 1970. The further studies on the separation of responsible protein in the skin test antigen FST by column chromatography, disc electrophoresis, and isoelectric focusing technique, p. 169-190. In, M. Sasa (ed.), Recent Advances in Researches on Filariasis and Schistosomiasis in Japan. Scott, D.W. and R.K. Gershon. 1970. Determination of total and mercaptoethanol-resistant antibody in the same serum sample. Clin. Exp. Immunol. 6:313-316. Scott, D.W., B.R.H. Farrow, and R.D. Shulta. 1974. Studies on the therapeutic and immunologic aspects of generalized demodectic mange in the dog. J. Am. Anim. Hosp. Assoc. 10:233-244. Scott, D.W., R.D. Schulta, and E. Baker. 1976. Further studies on the therapeutic and immunologic aspects of generalized demodectic mange in the dog. J. Am. Anim. Hosp. Assoc. 12:203-213. Shimp, R.G., R.B. Crandall, and C.A. Crandall. 1975. Ueligmosomoides polygyvus {Nematospiroid.es Jubius): Suppression of antibody response to orally administered sheep erythrocytes in infected mice. Exp. Parasit. 38:257-269. Simpson, C.R., B.M. Gebhardt, R.E. Bradley, and R.F. Jackson. 1974. Glomeruloclerosi s in canine heartworm infection. Vet. Pathol. 11:506-514. Smith, D.H. 1971. Evaluation of the Dirofilaria iivmitis filarial skin test antigen in the diagnosis of filariasis. WHO Bull. 44:771-782.

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Spira, D.T., J. Golenser, and I. '^ery. 1976. The reactivity of spleen cells from malarious rats to nonspecific mitogens. Clin. Exp. Immunol. 24:139-145. Steel, R.G.D. and J.H. Torre. 1960. Principles and procedures of statistics. McGraw Hill Book Co., New York, NY Takahashi, J. and K. Sato. 1976. Studies on hemagglutination test on filariasis. I. The fractionation and purification of antigens by column chromatography and disc electrophoresis. Japan. J. Exp. Med. 43:353-355. Thilsted, J. P. and M. Shifrine. 1977. Lymphocyte transformation in the dog: Response of lymphocytes from normal and immune dogs to phytohemagglutinin, coccidiodin, and purif ied-protein derivative. Am. J. Vet. Res. 38:81-87. Underwood, D.C. and P.D. Harwood. 1939. Survival and location of the microfilariae of Dirofilaria immitis in the dog. J. Parasit. 25:23-33. Vaitukaitis, J., J.B. Robbins, E. Nieschlag, and G.T. Ross. 1971. A method for producing specific antisera with small doses of immunogen. J. Clin. Endoc. 33:988-991. Voller, A., D. Bidwell, and A. Bartlett. 1976. Microplate enzyme immunoassays for the immunodiagnosis of virus infections. Am. Soc. Microbiol. Chap. 69:506-515. Weber, K. and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sul fate-polyacrylamide gel electrophoresis. J. Biol. Chem. 255:4406-4412. Weiner, D.J. and R.E. Bradley. 1970. A new modification of Knott's method for counting microfilariae. ASB Bull. 17:69. Weiner, D.J. and R.E. Bradley. 1972. Serologic changes in primary and secondary infections of beagle dogs with Dirofilaria immitis. p. 77-86. ]n, R.E. Bradley and G. Pacheco (eds.), Canine Heartworm Disease: The Current Knowledge. University of Florida, Gainesville, Florida. Weiner, D.J. and R.E. Bradley. 1973. The 2-mercaptoethanol labile immunoglobulin response of beagles experimentally-infected with Dirofilaria immitis. J. Parasit. 59:696-700. Wheeling, C.H. and W.F. Hutchison. 1971. Disc electrophoresis and Immunoelectrophoresis of soluble extracts of Dirofilaria immitis. Japan. J. Exp. Med. 41:171-176. Whitlock, H.V. 1948. Some modifications of the McMaster helminth egg counting technique and apparatus. J. Coun. Sci. Indus. Res. 21 : 177-1 80.

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Williams, J.R. and A.W. Dade. 19/6. Dirofilaria immitis infection in a wolverine. J. Parasit. 62:174-175. Winter, A., H. Perlmutter, and H. Davies. 1975. Preparative flat-bed electrofocusing in a granulated gel with the LKB 2117 niultiphor. Technical Communication, LKB Produkter AB, Stockholm, Sweden. Wong, M.M. 1964. Studies on microfilaremia in dogs. II. Levels of microfilaremia in relation to immunologic responses of the host. Am. J. Trop. Med. Hyg. 13:66-77. Wong, M.M. 1974. Experimental dirof i 1 ariasis in macaques. Susceptibility and host responses to Dirofilaria immitis, the dog heartworm. Trans. Roy. Soc. Trop. Med. 68:479-490. Wong, M.M., P.F. Suter, E.A. Rhode, and M.F. Guest. 1973. Dirofilariasis without circulating microfilariae a problem in diagnosis. J. Am. Vet. Med. Assoc. 163:133-139. Wong, M.M., M.F. Guest, and M.J. Lavoipierre. 1974. Dirofilaria immitis: Fate and immunogenic!' ty of irradiated infective stage larvae in beagles. Exp. Parasit. 35:465-474.

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BIOGRAPHICAL SKETCH Robert Burton Grieve was born October 27, 1951, at Torrington, Wyoming. He graduated from Lingle High School, Li ngle, Wyoming, in May, 1969. In September, 1969, Mr. Grieve initiated studies at the University of Wyoming, Laramie, where he received the Bachelor of Science degree in Microbiology in May, 1975. He worked as a diagnostic parasitologist at the Wyoming State Veterinary Laboratory from 1973 to 1974 while continuing graduate studies at the University of Wyoming. In May, 1975, he completed the Master of Science degree in Microbiology with the thesis in veterinary parasitology. Mr. Grieve enrolled at the University of Florida in September, 1975, to work toward the Doctor of Philosophy degree. He has held a graduate assistantship in the College of Veterinary Medicine (Institute of Food and Agricultural SciencesAnimal Research Facility) where he studied immunology and parasitology. Mr. Grieve is a member of the Alpha Zeta Fraternity, the American Association of Veterinary Parasitologists, the American Society of Parasitologists and the Helminthological Society of Washington. 99

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy./^ ' Richard E. Bradley, Sr., Chairman Professor, College of Veterinary Medicine I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. .IA1 n ,-tu 'Wl, Paul T. Cardeilhac Professor, College of Veterinary Medicine I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. L ( Harvey L. Cromroy Professor of Entomology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. / (_ #v( 7), W\ Edward M. Hoffmann Professor of Microbiology yand Cell Science

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This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. March, 1978 1,/, Tf 'I Dean,/ College of Agriculture Dean, Graduate School

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