I
u— .- >“l —

IMMUNCSDEFEUSEON STUDEES WETH
ASCARIS SUUM GOEZE, 1732 AND
TOXOCARA CANIS (WERNER), 1782

 

Thesis Ear the Degree 4:; pk. D.
MICHIGAN STATE UNIVERSKTY

Reginakd George Nash
1964

snags

LIBRARY

Michigan State
University

 

This is to certify that the

thesis entitled
Iifl-‘IUNODIFFUSION STUDIES WITH ASCARIS S___UUM

GOEZE, 1782 AND TOXOCARA CANIS
(WERNER), 1782

 

presented by

Reginald George Nash

has been accepted towards fulfillment
of the requirements for °

Ph. D.

degree in M

 

 
    

‘I' . Way‘ne Porter professor

Date November 25, 1964

0-169

ABSTRACT

IMMUNODIFFUSION STUDIES WITH ASCARIS SUUM

 

GOEZE, 1782 AND TOXOCARA CANIS

 

(WERNER), 1782

by Reginald George Nash

The antigens of Ascaris suum and Toxocara canis were

 

 

studied by a slide microtechnique of double diffusion. Diffusion
of antigens against homologous antisera produced in rabbits
indicated the following numbers of antigenic components in
various stages of the life cycles of the two species: six in the
unembryonated eggs of both species, four in the embryonated

eggs of both species, eight in the adult stage of _T_. canis. No

 

antiserum was used to the antigens of the adult stage ofé. suum.

 

Cross reactions were demonstrated by diffusion of each
antiserum against the heterologous antigens. The results in-
dicated that most antigenic components cross reacted with the
heterologous species.

Two specific antigens were demonstrated in the adult
stage of I. _c_£_i_r_1'i_s when compared to the adult stage of 9:. s_u1_1_r_r_1.

Absorption of the antiserum to adult '_I‘_. canis with the antigen to

Reginald Geor ge Nash

adult A. suum and diffusion of the absorbed antiserum against
the antigen of adult I. canis produced two precipitin lines.
These two precipitin lines represent specific antigens of I.

canis.

 

Absence of mutual absorption of antisera with antigens
indicated a greater complexity of antigenic components in un-
embryonated eggs, embryonated eggs and whole worms of A.

suum and I. canis than was revealed by diffusion of antisera

 

 

to each of the stages in the life cycles of these roundworms
against their homologous antigens .
Precipitins were indicated in the serum of mice infected

with A. suum or T. canis when the serum of infected mice was

 

 

diffused against microcultures of living larvae of the species
with which the mice were infected.
The effects on the serum of albino mice infected with

A. suum or T. canis were investigated by immunoelectrophore-

 

 

sis. Twenty serum fractions, including two alpha -twos not
reported by previous investigators, were demonstrated in
normal mouse serum with this technique. Comparison of the
immunoelectrophoretic patterns of infected and normal mice
revealed that there was an increase in (2211 globulin in mice
infected with A. w and an increase in gamma globulin in

the serum of mice infected with I. canis. Dilutions of the sera

 

Re ginald George Nash

of infected mice prior to electrophoresis compared with sim-
ilar dilutions of normal mouse serum proved that the apparent
increases in globulin fractions in infected mice were not due
to maintenance of the levels of (12 II and gamma globulins while
other components of the serum decreased in amount.

The increase in the (12 II globulin in mice infected with

A. suum and the increase in gamma globulin in mice infected

 

with I. canis were not due to production of antibodies to anti-

 

gens in the unembryonated eggs, embryonated eggs or adults
of the species with which the mice were infected. Absorption
of the sera of infected mice with these antigens had no effect
on the increased fractions in the sera of infected mice.

The changes in serum globulin fractions of mice infected

with A. suum or T. canis are not specific to the roundworms.

 

 

The increase of (1‘2 11 globulin in mice infected with A. suum

 

may be the result of inflammation and tissue damage in the

liver since the larvae of A. suum remain in the liver much

 

longer than those of I. canis. The increase in gamma globulin
in the serum of mice infected with _T_. canis may be due to bac-
terial invasion of the tissues of the intestine following penetra-

tion of the larvae.

IMMUNODIFFUSION STUDIES WITH ASCARIS SUUM

 

GOEZE, 1782 AND TOXOCARA CANIS

 

(WERNER), 1782

By

Re ginald George Nash

A THESIS

Submitted to
Michigan State University
’ in partial fulfillment of the requirements
for the degree of

DOC TOR OF PHILOSOPHY

Department of Zoology

1964

 

ACKNOWLEDGMENTS

Sincere gratitude is extended to Dr. T. Wayne Porter,
Dr. David T. Clark and Dr. Rollin H. Baker for their excep-
tional guidance and assistance during the course of this
investigation.

Acknowledgment is given to the Department of Zoology
and to the Department of Microbiology and Public Health for
providing the equipment and materials needed to complete
this study.

The efficient attention to details by Mrs. M. Bernadette
Henderson has been greatly appreciated.

Special thanks is given to Mr. R. D. Van Deusen for
providing laboratory facilities at the W. K. Kellogg Bird
Sanctuary.

The assistance of my wife has been invaluable in many

phases of this study for which I am deeply grateful.

ii

TABLE OF CONTENTS

Chapter Page
INTRODUCTION ................................... 1
RELATED LITERATURE ........................... 4
MATERIALS AND METHODS ....................... 27
A. Gel diffusion studies ...................... 27
B. Immunoelectrophoretic studies ............. 35
RESULTS ......................................... 43
A. Gel diffusion studies . . . . . ................. 43
B . Immunoelectrophoretic studies ............. 55
DISCUSSION ....................................... 67
A. Gel diffusion studies ...................... 67
B. Immunoelectrophoretic studies ............. 71
SUMMARY AND CONCLUSIONS ........... . . . . ...... 78
A. Summary ............. . ......... 78
B. Conclusions . . . . ........................ . . 80
BIBLIOGRAPHY .................................... 84

iii

Table

 

LIST OF TABLES

Page

Numbers of precipitin lines developed
when antisera are diffused against homol-
ogous and heterologous antigens ..... . . . . . . . 46

Numbers of precipitin lines detected

when antigens are diffused against

antisera A and C absorbed with each

antigen .......... . ....................... 49

Numbers of precipitin lines observed

when antigens are diffused against

antisera D and E absorbed with each

antigen ....... . . . . .......... . ............ 52

Numbers of precipitin lines demon-

strated by diffusion of antigens against
antiserum F absorbed with each antigen ..... 53

iv

Figure

LIST OF FIGURES

Page
Gel Diffusion Slide Illustrating
Configuration of Wells for Reactants ......... 31
Composite of Immunoelectrophoretic
Patterns of Normal Mouse Serum ........... 58
Alpha -two Components in Normal Mouse
Serum .......... . ............. . .......... 59
Immunoelectrophoretic Patterns of
Undiluted Mouse Serum . ..... . . . . . . . . ..... . 61
Immunoelectrophoretic Patterns of
Diluted Mouse Serum ..... . . . . . . . . . . . ...... 63
Immunoelectrophoretic Patterns of
MouseSerum ........ ....... 65
Immunoelectrophoretic Patterns of
Mouse Serum . . . . . .............. . ......... 66

INTRODUCTION

A comparative study of the antigens of Ascaris suum

 

Goeze, 1782 and Toxocara canis (Werner), 1782 was completed

 

during the course of this investigation. The double diffusion
slide microtechnique of Wadsworth ( 1957) was used.
Characterization of the antigens of parasitic helminths
has been a subject of frequent investigation due to their in-
volvement in most immunological responses of the host includ-
ing immunity and hypersensitivity. Antigens which are specific
to helminths and others which are common to many organisms
have been demonstrated. Kent ( 1960) isolated water soluble
antigens from A. s_ugr_n, four of which were shown to be quite
specific by precipitation with homologous antibodies from ex-
perimentally infected rabbits and by lack of cross reactivity
with sera from other helminth infections such as trichinosis,
echinococcosis and schistosomiasis. The Forssman antigen,
present in widely separated species, was demonstrated in Trichin-

ella spiralis by Mauss ( 1941) . Marked elevation in heterophile

 

antibodies against the Forssman antigen was demonstrated in

animals infected with A. suum by Soulsby ( 1958). Oliver-

Gonzales ( 1946) reported that rabbits infected with A. suum

 

showed an increase in the A1 and A2 blood isoagglutinins.

Antigens that stimulate the production of antibodies which
do not induce an immunity that prevents re-infection are called
nonfunctional antigens; those which stimulate the production of
antibodies active in immunity are called the functional antigens
(Soulsby 1963) . The isolation of specific antigens is of im-
portance in relation to diagnostic tests but specific antigens
can not be considered as synonymous with functional antigens
(Soulsby 1963) .

Analyses of the antigens of A. w by gel diffusion
methods have been reported by Soulsby ( 1957) and Kagan

( 1957) . Huntley and Moreland ( 1963) demonstrated the sim-

 

ilarity between the antigens of Toxocara canis and A. lumbri-
coides by use of gel diffusion.

Recent investigations by means of fluorescent antibody
techniques indicate that the functional antigens are elaborated
by larvae of the parasitic nematodes (Taffs and Voller 1963;:
Crandall e_t a_1. 1963).

A study of the effects on the serum of albino mice infected
with A. s_uu_rn or _T_. e_anls was undertaken in this study by use

of the micromethod of immunoelectr0phoresis developed by

Scheidigger ( 1955) .

The alterations in serum proteins during infection with
several nematode parasites have been investigated. Leland
e_t 32' (1959) demonstrated that in calves infected with Tricho-

strongylus axei there was a decrease in total serum proteins

 

with an increase or maintenance of the alpha -two globulin level.
Andersen e_t a_l. ( 1960) reported that calves infected with the

medium stomach worm, Ostertagia ostertagi, showed a marked

 

decrease in amount of albumin and an increase in one or more
of the globulins. Leland e_t fl. ( 1955) found that in rats in-

fected with Nippostrongylus muris the total serum protein was

 

slightly increased and the beta globulin was distinctly increased.
Weber ( 1957) found the gamma globulin was increased in cattle

exposed and re -exposed to the lungworm Dictyocaulus viviparus .

 

There was an increase in beta and gamma globulins in lambs

infected with Strongyloides papillosus as reported by Turner ( 1959) .

 

Sadun ( 1957) demonstrated that rabbits infected with Toxocara
canis developed hypergammaglobulinemia and there was an in-

crease in the alpha -one fraction of the serum.

RELATED LITERATURE

There are several methods of gel diffusion: single dif-
fusion described by Oudin ( 1952) , double diffusion devised
by Ouchterlony ( 1953) , and immunoelectr0phoresis developed
by Grabar and Williams ( 1953) .

Oudin ( 1952) , of the Pasteur Institute, developed the
first method of gel diffusion which consisted of capillary tubes
filled with agar in which complex antigen-antibody systems
could be analyzed by allowing reactions to occur. The anti-
bodies were mixed with the agar which was then allowed to
harden. Antigens were overlaid and layers of precipitates
formed at different levels depending upon the rates at which the
antigens diffused into the gel. This method is known as simple
or single diffusion.

Ouchterlony ( 1953), at the Karolinska Institute, developed
a similar method in which both the antigen and antibody were al-
lowed to diffuse into an agar plate which initially contained

neither reactant. Because of this,~ the method was called double

diffusion. Drops of antigen and antibody were placed in sep-
arate wells cut in an agar plate. Movement of the reactants in
the agar depended upon their concentrations and diffusion co-
efficients. Precipitin lines formed when the diffusing antigen
met its specific antibody or antibody with which it could cross
react. Identification of these precipitin lines was possible if
either a known antigen or antibody was placed in a third well.
When the diffusing reactants met, the line of precipitate formed
by this reaction joined the line formed by the previous reaction
to form a continuous precipitin line. Naming of various precipitin
lines as reference points in a complex mixture of proteins was
possible by this method.

Immunoelectrophoresis, the most recent of the methods
in gel diffusion to be developed, is based upon the principle of
electrophoresis which has long been known. Electrophoresis
may be defined as the migration of particles under the influence
of an electrical field (Lewis, 1960) . Porret (1816) observed the
passage of water through an animal membrane under the in-
fluence of an electrical current. Hardy ( 1899) found that
negatively charged particles of egg albumin could be made posi-
tive by addition of acid and with the change from alkalinity to
acidity the particles migrated in the opposite direction in the

electrical field. The electrical properties of proteins were

I,

determined by the nature of the reaction, either acid or alka-
line, in the field. If the field was alkaline the particles became
electronegative or if the field was acid the particles became
electropositive. The particles moved at different speeds
depending upon the net charges of the particles -_i_.g. , the
greater the number of charges, the faster the migration (Lewis
1960) .

The microscopic method of electrophoresis was first
used to study blood cells, protozoans and bacteria in a liquid
medium in which ionizing compounds were incorporated to
render it conductive (Abramson 1934) . The medium contain-
ing the electrolytes and the cells to be studied were put into
a glass tube and placed horizontally on the microscope stage.

A current was then passed through the tube and the movements
of the blood cells, protozoans and bacteria in the electrical
field could be observed.

The microscopic method was replaced by the moving boun-
dary method of electrophoresis perfected by Tiselius ( 1937) .
Movement of the boundary between two solutions such as a. pro-
tein and a buffer could be observed if an electrical current was
passed through the two solutions after they were placed in a
U-shaped tube so a sharp boundary existed between the two. A

series of moving boundaries representing different protein ions

O—Oo

was obtained upon passage of an electrical current through
the tube when the protein solution contained ions of different
charges or sizes. By this method Tiselius (1937) discovered
that the serum globulin of the blood contained three fractions
which he named alpha, beta and gamma globulin.

The late nineteen forties marked the development of a
third technique called zone electrophoresis (Kunkel and Tiselius
1951) . In this method a solid or semi-solid medium was used
through which the electrically charged particles were moved by
an electrical current. The separated particles aggregated in
zones in the medium. Thus it was known as zone electrophore-
sis. By 1950 this method was being used in diagnosis of
nephrosis, liver malfunction and multiple myeloma.

Grabar and Williams ( 1953) reasoned that it might be
possible to combine the refinement of separation of protein
mixtures by electrophoresis with the double diffusion technique
of Ouchterlony. If a glass plate were covered with buffered
agar gel and the serum were electrophoresed in the agar gel,
then the various proteins in the serum should be strung out in
a line of spots or zones as if they had been placed there in the
first place. After electrophoresis the antigens would diffuse
radially from each of the spots as they would if on an Ouchter-

lony plate. In order to locate these antigens visually, antibody

solution could be placed in a longitudinal trough on the plate
and allowed to diffuse laterally toward the radially diffusing
spots of antigen. When the specific antibody and antigen
encounter one another a precipitin line should form in an arc.
This technique is now known as immunoelectr0phoresis.

The method of immunoelectr0phoresis offers great
refinement in the separation of protein mixtures (Crowle
1961) . Older methods such as chemical analysis of groups
of proteins yielded the separation of albumin and globulins.
The globulin fraction can be separated into alpha, beta and
gamma globulin by zone electrophoresis as stated above.
These fractions are also made up of groups of proteins as
revealed by immunoelectrophoretic studies of serum (Williams
and Grabar 1955) . The technique of immunoelectr0phoresis
as described by Grabar and Williams ( 1953) required rather
large amounts of reactants and a long period of electrophore-
sis. Scheidigger ( 1955) adapted the procedure to a miniature
method which made it possible to perform the entire procedure
on a microscope slide covered with a very thin layer of buf-
fered agar gel. This greatly shortened all steps of the procedure
and also reduced the amounts of reactants needed.

Clausen and Heremans ( 1960) introduced a further mod-

dification of the procedure by which it was possible to demonstrate

 

 

.( D

identical reactions in two samples electrophoresed on the same
slide, very much in the same way these reactions can be
identified on an Ouchterlony plate. Agar was aspirated from
an agar covered slide to produce two circular holes used as
wells for the antigens, eight millimeters distance being left
between the two wells. A discontinuous or interrupted trough
was placed between the two antigen wells and a continuous
trough was placed laterally to each of the antigen wells. Fol-
lowing electrophoresis of the sera the lateral troughs were
filled with the respective homologous antisera and the central
discontinuous trough was filled with a mixture of the two anti-
sera. Identical precipitins from both of the electrophoresed
sera formed continuous arcs.

Previous to 1955 only three papers existed relating to
the use of electrophoresis as a tool to elucidate the effect
of helminthic infections on the plasma proteins of the host.
These are by Wright and Oliver-Gonzales ( 1943) and Schwonzen

(1951) concerning Trichinella spiralis and Evans e_t _a_1. (1955)

 

on Schistosoma mansoni.

 

Since 1955 numerous papers have appeared in the litera-
ture concerning changes in plasma proteins as the result of

helminthic infection. Leland, e_t a_1. ( 1955) reported that in

10

rats infected with Nippostrongylus muris the total serum pro-

 

tein was slightly increased and the beta globulin was greatly

increased. In rats heavily infected with Cysticercus fasciolaris,

 

Kraut ( 1956) found there was an increase in total globulin due
to increases in the alpha-two, beta and gamma globulins. He
concluded that these changes were not specific and that the
degree of increase in globulins was correlated with the degree
of liver involvement. Ground worm material was injected

into rats and an immunity to subsequent infection developed
without increases in any globulin fraction. Sadun e_t .a_l. ( 1957)

demonstrated that rabbits infected with Toxocara canis developed

 

hypergammaglobulinemia and there was an increase in the
alpha-one fraction of the serum. Weber (1957) found that
gamma globulin was increased in cattle exposed and re-exposed

to the lungworm Dictyocaulus viviparus. An increase in the

 

beta and gamma globulins in lambs infected with Strongyloides

 

papillosus was reported by Turner (1959) . Leland gt _a_l. (1959),

studying Trichostrongylus axei infections in calves, observed

 

hypoproteinemia and an increase or maintenance of the alpha-
two globulin level. Kruidenier and Katoh ( 1959) observed an
increase in the beta and gamma globulins in the serum of cats

and rats infected with the lung fluke Paragonimus kellikoti.

 

Andersen _e_t a}. ( 1960) demonstrated that calves infected with

11

the medium stomach worm, Ostertagia ostertagi, showed a
marked decrease in the amount of albumin and an increase in
amount of one or more of the serum globulins.

Stauber ( 1954) , basing his statement largely on chemical
analyses, reported that albuminemia and hyperglobulinemia are
characteristic of many conditions including parasitic infections.
Loss of plasma protein resulting in albuminemia could be due
to lesions in the alimentary tract through which leakage of
albumin could occur (Leland 1961) . Perlmann e_t fl. ( 1943)
found that with lesions caused by burns there was a decrease
in total plasma. volume. Changes in the relative amounts of
various serum proteins may be the result of rate of synthesis
or loss of some fractions by internal destruction or external
leakage (Leland 1961) . There is a possibility that a decrease
in albumin which usually maintains serum osmotic pressure
may effect an increase in some other serum fraction to com-
pensate for the loss (Leland 1961).

The liver was indicated as the site of production of serum
albumin by Peters and Anfinsen ( 1950) . The alpha and beta
globulins are also thought to be synthesized in the liver (Leland
1961). Miller and Bale ( 1954) found that the alpha globulins

(were produced and turned over at a more rapid rate than other i

12

protein components of the serum. Raffel ( 1961) believes
that plasma cells are the site of production of gamma globulin.

Kuttler and Marble ( 1960) produced an experimental
anemia in sheep by bleeding them in order to determine if the
changes observed in serum fractions during parasitic infec-
tions could be due to the anemia which frequently accompanies
such infections. The results indicated that the changes in
serum proteins observed during infection were not due to
anemia.

Shedlovsky and Scudder ( 1942) found that an increase
in alpha globulin occurred when inflammation or tissue damage
was involved. The changes in serum proteins of hosts infected
with parasitic helminths are associated with inflammation or
tissue damage and therefore the changes can not be considered
as specific to the parasite (Leland 1961) . Further evidence to
support the view that changes in serum globulin fractions during
infection with parasites are non-specific was found in the work
of Atchley e_t a_l. ( 1961) in regard to experimental infection of

guinea pigs with the protozoan Entamoeba histolytica. Heavily

 

infected animals developed a change in the alpha -one region as
revealed by immunoelectrophoretic technique. Since tissue
damage should be correlated with the degree of infection, the

change in the alpha -one region could very well be associated

13

with the extensive tissue damage present in heavy infections with

E. histolytica.

 

Kent ( 1960) isolated antigens from adult female A. m.
The worms were homogenized and defatted with ethanol and
ether and extracted with buffered distilled water. After cen-
trifugation the decanted supernatant was used as a source of
antigens. The proteins were separated by paper electrophore-
sis and agar gel electrophoresis. Five major fractions were
recovered. Ouchterlony double diffusion tests and immuno-
electr0phoresis indicated at least four specific antigens pre-
cipitated by the homologous antibodies from infected rabbits .

Sera from suspected cases of visceral larva migrans, presumably

of Toxocara canis origin, did not react with the antigens in most

 

cases but the sera of the two human patients with A. lumbri-
coides infections reacted with the antigens twelve days before
eggs were found in the stools.

The immunoelectrophoretic characteristics of normal mouse
serum was presented by Heremans 31: El" (1959) . Antimouse
serum containing the largest number of antibodies was produced
by giving rabbits four intramuscular injections of O. 5 milli-
liter of mouse serum at intervals of six weeks. Freund's
adjuvant was used with the first of these injections . Immuno-

electrophoresis was done by the methodof Scheidigger ( 1955) .

14

After development of the precipitin arcs, the bows were
labeled according to their relative electrical mobilities within
the alpha, beta and gamma regions, starting with those most
anodically placed and progressing to those most cathodically
placed. Heremans e_t a_l. (1959) observed at least twenty dif-
ferent fractions in mouse serum. These were a pre-albumin,
albumin, four bows in the alpha -one region, six in the alpha-
two region, one in the beta—one region, four in the beta-two
region, three in the beta -three region and one in the gamma
region.

Later, in the immunoelectrophoretic studies of sera
from mice carrying transplantable plasma cell leukemias,
Clausen and Heremans ( 1959) found that increases in amounts
of various fractions of the serum could be ascertained by a
dilution of serum previous to electrophoresis. Comparisons
of the dilutions of serum from normal animals with the serum
of animals bearing the transplantable plasma cell leukemias
revealed changes which were not readily detectable without
dilutions of the sera.

The similarities of human and mouse serum proteins
were demonstrated by Clausen and Heremans ( 1960) by use
of the immunoelectrophoretic method. Two antigen wells

were placed on an agar covered slide. Mouse serum and

15

human serum were placed in the two respective wells. A
trough parallel to the path of electrophoretic migration was
cut laterally to each of the antigen wells into which the
homologous antisera were placed following electrophoresis.
Another discontinuous or interrupted trough was cut between
the antigen wells into which a mixture of antihuman and anti-
mouse antisera was placed. This way of cutting the agar left
several areas through which proteins of both mouse and human
sera could diffuse toward one another. The method allowed
cross reactions to occur between homologous mouse and human
proteins. Mouse serum proteins which cross reacted with
horse antihuman antiserum were prealbumin, albumin, four
alpha globulins, three beta globulins and gamma globulin.
Among the fractions of mouse serum which were found to
share antigenic determinants with human serum were gamma
globulin and call globulin, found to be related to human haptoglobin.
There has been considerable temptation to interpret any
changes in serum protein fractions of hosts infected with hel-
minth parasites as being associated with immunity but there
has been little justification for this except when the gamma
globulin fraction was involved (Leland 1961) . Immunity of
a host to helminth parasites is the result of response of the

host to antigens of the parasite. Consequently, the antigens

16

of helminths have been investigated to determine which antigens
are involved in the production of immunity of the host (Soulsby
1963) .

The antigens of ascarids have been investigated fre-
quently since Canning ( 1929) demonstrated that various tis-

sue fractions of A. suum were antigenic. He injected rabbits

 

with various tissues of A. suum to obtain antisera. Higher

 

titres of antibody were secured with homologous tissue antigens
than with heterologous tissue antigens. The antigenicity of a

polysaccharide fraction from A. suum was demonstrated by

 

Campbell ( 1936) . In 1937 he found that the polysaccharide
fraction of ascarids of humans and pigs were ser'ologically
specific. Oliver-Gonzales ( 1943) reported that the egg of
A. M contained an antigen responsible for precipitins in the

serum of immune rabbits which killed the larvae of A. suum in

 

vitro. In 1944 he found that the polysaccharide fraction of A.

suum inhibited agglutination of human erythrocytes and that

 

the polysaccharide fraction was related immunologically to
human erythrocyte antigens in type A and type B blood. The
polysaccharide fraction inhibited the hemolysis of sheep cells.

He concluded that the polysaccharides of A. suum and other

 

helminths were antigenic and related to human isoagglutinins

and Forssman antigen. In 1946 he found that infection in

17

rabbits with A..s_u_1_1_rr_1 larvae caused an increase in A 1 and
A 2 isoagglutinin titre in the hosts serum. Serum absorbed
with cuticle of A. s_1i1_1_r_n gave no reduction in titre. In 1946
he showed that helminth polysaccharides inhibited the action
of A 1 and A 2 isoagglutinins in human serum. In 1953 he
reported that the erythrocytes in type O and type B human

blood could absorb the polysaccharide fraction of A. suum.

 

The A 2 isoagglutinin titre of serum was reduced to zero with
these antigens.
Sprent and Chen ( 1949) found that active or passive

immunity could not be induced in mice with whole worms or

various tissue antigens of A. suum.

 

Sprent ( 1949) found that repeated intravenous injections
of antigens of A. m in humans, rabbits, guinea pigs and mice
produced skin sensitivity in the absence of precipitins in man,
anaphylactic sensitivity in rabbits and precipitins in the absence
of skin sensitivity in mice. In 1950 he found that guinea pigs in-
fected with A. w could be anaphylactically shocked with other
nematode antigens but not with pneumococcus polysaccharide, hemol-
yzed group A erythrocytes, normal guinea pig kidney and lung, acantho-
cephala, cestode, annelid or molluscan antigens. The substance caus-

ing shock was both protein and polysaccharide in nature. In 1951 he

18

reported that guinea pigs infected with A. suum could be ana-

phylactically shocked with extracts of the larvae of A. suum

 

and their metabolic products .

In reporting on the behavior of the larvae of A. m in
mice, Sprent ( 1952) stated that the larvae that penetrated the
intestine went to the liver, lungs and intestine from which they
were expelled with the fecal material. On the way to the lungs
the larvae grew from an average of 0.2 mm. to 1.05 mm. in
length in a period of eight days . In mice that were reinfected
few larvae migrated to the lungs and those that did were much
smaller than those in the first infection. Many larvae were
encapsulated by accumulations of inflammatory cells. Sprent

( 1952) reported that in mice infected with A. suum the larvae

 

stayed in the liver for four days following infection. Congestion
of the lungs appeared from the fifth through the eighth day.

No larvae were found in the mice after two weeks. The larvae
of 2. 9.3915 in mice were found to migrate through the somatic
tissues without reaching the intestine. The kidneys were badly
infected from the second to the seventh day. The larvae left

the liver much sooner in mice infected with I. canis than in

 

mice infected with A. suum.
Nichols ( 1956b) stated that the behavior of the larvae of

T. canis and A. suum was strikingly similar in mice and man.

19

Beaver ( 1952) called this condition visceral larva migrans.

Heiner and Kevy ( 1956) found that if I, canis was the causative

 

agent the condition persisted for as long as sixteen months and
with massive infections death resulted. Nichols (1956a) re-
ported that the only means of diagnosing this syndrome was to
find the larvae in the tissues of man. Differential serological
diagnosis may be possible in the future by isolation of specific
antigens from the larvae of each of the species (Kagan e_t _a_1.
1959) . Cross reactions between the antigens of A. m and

T. canis preclude serological diagnosis at present (Huntley

and Moreland 1963). The analysis of antigens of A.§1_1_ui_n by
double diffusion was reported by Soulsby ( 1957) . Antiserum in
0. 4 percent agar was placed in a serological tube, overlaid

by 0. 5 milliliter of 0. 4 percent agar in saline and superimposed
by 0. 5 milliliter of antigen homologous to the antiserum. The
antigens used were saline extracts of the tissues of A. m.
Double diffusion was allowed to procede for fourteen days. Whole
worm antigen produced a maximum of nine bands of precipitate
with its homologous antiserum; intestine produced fourteen bands;
cuticle produced nine bands. Dense rings indicated protein anti-
gens whereas fine rings indicated polysaccharide antigens.

Kagan ( 1957) reported the results of double diffusion

20

studies with antigens of A. suum. Antisera against whole worm
antigen was produced in rabbits. Antigens were prepared from
unembryonated eggs, embryonated eggs, enteric fluid, muscle,

cuticle and whole worms of A. suum. Whole worm antigens were

 

prepared from '_I‘_. canis, _T_. cati and A. lumbricoides, ascarids

 

 

from the dog, cat and man respectively. Double diffusion tests
indicated there were ten antigenic components when whole worm
antigen of A. m was tested against full strength antiserum.

Full strength antiserum to whole worm antigen of A. m in-
dicated the following number of antigenic components when

tested against various tissues: muscle, five; cuticle, six; em-
bryonated eggs, two; unembryonated eggs, sixf. When the full
strength antiserum to whole worms of A.fl1_1_1_r_n was diffused against

whole worm antigen of T. canis, four bands of precipitin were

observed; with I. cati, five; with A. lumbricoides, seven.

#7

 

In all of the absorption studies done by Kagan ( 1957) , the

antiserum to whole worms of A. suum was diluted one to four

 

which greatly simplified the results since many precipitin lines
did not appear with this dilution of the antiserum. This procedure
does not reveal whether the precipitin lines lost by dilution of the
antiserum would have been absorbed. When the diluted anti-
serum to whole worms of A. wwas diffused against the homol-

ogous antigen four precipitin bands were observed. When the

21

diluted antiserum was absorbed with the antigen of unembryonated
eggs of A..s_1£1_r_n, no precipitin lines were observed. Absorption
of the diluted antiserum with antigen of embryonated eggs and
subsequent diffusion against whole worm antigen yielded four
precipitin lines. These results indicated that the unembryonated
egg contained all of the antigens present in the adult stage but
the embryonated egg contained none of the antigens present in
the adult stage.

Huntley and Moreland ( 1963) demonstrated the antigenic

similarity between T. canis and A. lumbricoides, ascarids from

 

the dog and man respectively, by use of a gel diffusion technique.
An antiserum produced in rabbits to antigens in whole worms of
I. canis was used throughout the study. Fresh antigens from

adult whole worms of I. canis and A. lumbricoides were diffused

 

against the antiserum. At least nine antigenic components were

demonstrated in the extract from whole worms of _'_I‘_. canis and

 

at least five cross reacting antigens were observed in the extract

from the whole worms of A. lumbricoides.

 

Cross reactions between closely related ascarids have been
noted by many: Kagan e_t_z_:1_l_. ( 1959), using the bentonite floc-
culation test; Olson ( 1960) , using the in vitro larval precipitate

test; Huntley and Moreland (1963), using gel diffusion.

22

Recent investigations by me ans of fluorescent antibody
techniques indicate the antigens functional in immunity are
elaborated by larvae. Taffs and Voller ( 1963) reported that
the embryonated eggs of A. 21in were antigenic in the sense
that they stimulated the production of protective antibodies
by the host. The third stage larvae had antigenic sites at
the natural orifices and on the cuticle. Crandall e_t a_l. ( 1963),
working with A. su_ur_n, found by means of fluorescent tech-
niques that the cuticle of second and third stage larvae was
antigenic but that the cuticle of the adult stage was not.

A method of detection of metabolic antigens from nema-
tode larvae by a microculture agar gel technique was reported
by Olson :23 _a_l. ( 1960) . Excretions and secretions of living

larvae of Trichinella spiralis were allowed to diffuse into the

 

agar where reactions with sera of rabbits infected with the
nematode could be observed.

In all analyses by gel diffusion methods, results indicate
that molecules may carry determinant groups in exposed, reac-
tive positions or in hidden, non-reactive positions (Ouchterlony
1962) . He reported that most biological materials carry similar
molecules with determinants of more than one kind in various

combinations .

23

When two antigens are being compared by double dif-
fusion methods, precipitin lines in the patterns are charac-
terized by size, density, stability, whether stationary or
migratory, broad or narrow, sharp or diffuse, splitting or
coalesced (Ouchterlony 1962) . Precipitin lines form when
the reactants meet at equivalence. Diffusion coefficients
and initial concentrations of reactants as well as solubility
of the precipitate in antigen and/or antibody will determine
the site of precipitation. If the precipitin line is stationary
at the site of its first appearance, there is equivalence of
antigen and antibody at this site and at the source of origin.

Duplication of lines does not occur unless one reac-
tant is in excess of the other (Ouchterlony 1962) . He stated
that under such conditions the selective barrier of the precip-
itate is diminished and the reactant that is in excess may
diffuse through the barrier and cause a duplicate precipitin
line to be formed. Another possibility to account for duplicate
lines is that different determinants may be attached to the same
antigen molecule as suggested by Jennings e_t a_1. (1960) .

Ouchterlony ( 1962) stated that in comparative studies
of antigens it was necessary to consider the possibility that

the antigens are bispecific or multispecific, that is, the

24

reactive particle may carry determinants of two or more
separate specificities. Antibodies may also be bispecific
or multispecific.

Korngold ( 1956) stated that the concept of cross reac-
tion applied to serological relationships between different
single antigens which possessed similar structural groupings
within their molecules. An antiserum containing antibodies
against structural groupings or determinants that two different
proteins have in common will react with both proteins to the
same extent. If the antiserum contains antibody against a
grouping in the homologous antigen but absent from the cross
reacting antigen the latter will react with less antibody than
the former. Such an antiserum will still react with the homol-
ogous antigen after having been absorbed by the heterologous
antigen.

Two different mixtures of proteins are often compared
on an Ouchterlony double diffusion plate by diffusion of the
protein antigens against an antiserum that is homologous to one
of the protein mixtures (Korngold 1956) . The wells to contain
the reactants are usually placed so they form the corners of an
equilateral triangle. Precipitin lines which completely coalesce

will form if the antiserum contains antibodies against structural

25

groupings which the two different proteins have in common.
Precipitin lines bend toward one another at the point of
coalescence. Sometimes an antiserum contains antibody against
determinants that the homologous and cross reacting antigens
have in common as well as against determinants that are
unique to the homologous antigen. The precipitin lines formed
by reaction of the antiserum with the homologous and heterol-
ogous antigen will not coalesce perfectly but the precipitin
line formed by the homologous antigen will continue beyond
the point of union of the two precipitin lines to form a "spur".
Two different antigens are sometimes compared by
double diffusion against an antiserum that is homologous to
neither of the antigens (Ouchterlony 1962) . Double spurs can
form when an antiserum reacts with the two heterologous but
cross reacting antigens if these antigens have at least one
determinant that is common and one that is different. The
density of double spurs diminishes abruptly in relation to the
main precipitin lines. The two precipitin lines coalesce before
the double spurs appear and remain fused after the spurs develop.
Spurs can be used to determine the degree of cross reactivity
(Korngold 1956) . The antigens which are not closely related

give dense and greatly extended spurs. Closely related antigens

26

give short spurs which deviate from the direction an uninfluenced
line would have taken.

When two antigens are diffused against an antiserum on
an Ouchterlony plate precipitin lines often intersect without
coalescing in a reaction of non-identity (Ouchterlony 1962) . Re-
actions of non-identity can be distinguished from double spurs
because the density of precipitin lines does not diminish rapidly
beyond the point of intersection. Precipitin lines do not bend
toward one another at the point of intersection in reactions of
non-identity (Ouchterlony 1962) .

The wide application of diffusion-in-gel methods for im-
munological analysis is reflected in a bibliography published
by Ouchterlony (1962) in which there are 1185 references cited

in the field during the period from 1956 to 1960.

27

MATERIALS AND METHODS

A. Gel diffusion studies.

The adults of Toxocara canis were secured from eight to

 

twelve week old dogs obtained from various Humane Societies.
Piperazine citrate was used as an anthelmintic. The adult

females of I. gain—is were slit longitudinally and the distal

one -third of the bifurcated uterus was removed. Removal of the eggs
was accomplished by placing each uterus between two micro-

scope slides and applying pressure. The eggs were placed in

0. 5 percent formalin to inhibit bacterial and mold growth dur -

ing embryonation. The eggs were aerated by stirring daily

for a period of a month when it was assumed that all eggs

would have completed embryonation.

Because of the large numbers of embryonated eggs
required for these studies, round, covered plastic containers
eight inches in diameter were used so that the eggs from at
least fifteen adult female worms could be embryonated in one
container. After embryonation, eggs were stored at room

temperature .

28

The eggs in the proximal two thirds of the uteri were
removed by pressure and frozen for future use in preparation
of unembryonated egg antigen.

Occasionally, a few dogs were secured from private
sources. These dogs were euthanized after which the intestines

were stripped to remove the adults of I. canis. Adult female

 

worms were placed in saline and incubated at 370 C. for
seventy two hours during which oviposition continued. The
female worms were then removed and the eggs were separated
from the saline by centrifugation. The eggs were embryonated
in 0. 5 percent formalin. Large numbers of eggs were obtained
by this method but many did not embryonate . A higher per-
centage of embryonation was secured with eggs taken from the
uteri of worms removed from their hosts by anthelmintics.

The adults of A. suum were obtained by stripping the in-

 

testines of slaughtered pigs. Eggs were removed from the
uteri of the female worms by pressure and the eggs were em-
bryonated in the O. 5 percent formalin as described above for

T. canis. Unembryonated eggs were collected as described

 

above for I. canis and frozen for future use in preparing an
antigen.
Water soluble antigens were prepared from unembryonated

eggs, embryonated eggs and adult whole worms of each of the

29

nematode species. Unembryonated egg antigen was prepared
by placing equal volumes of unembryonated eggs and saline in
a homogenizer and by means of a Sargent's cone driven stirring
motor homogenization was attained in ten minutes. This
material was centrifuged at 4000 r.p.m. for ten minutes and
the supernatant was divided into two milliliter amounts and
frozen for future use as unembryonated egg antigen. Em-
bryonated egg antigen was prepared by removal of the eggs
by centrifugation from the formalin solution in which they had
been embryonated. The eggs were washed five times in dis-
tilled water to remove the residue of formalin. Equal
volumes of embryonated eggs and saline were homogenized
by the method described for the unembryonated eggs. After
centrifugation the supernatant material was divided into two
milliliter amounts and frozen for future use as embryonated
egg antigen. Whole worm antigen was prepared by homogeniz-
ing equal volumes of whole worms and saline. This material
was centrifuged for ten minutes at 4000 r.p.m. The super-
natant was divided into two milliliter amounts and frozen for
future use as a whole worm antigen.

An antiserum to each of the six antigens was produced.

Each of six rabbits was injected intraperitoneally twice weekly

30

for a period of two months with two milliliters of one of the six
respective antigens. After the initial series of injections of
antigen each of the rabbits was injected with three milliliters
of the appropriate antigen every month to maintain antibody
production. The rabbits were bled by intracardial puncture
nine days after the last injection of antigen. Rabbits could be
bled without anesthesia if they were placed on their backs with
the eyes covered. Fifteen milliliters of blood could be removed
each month from each rabbit without apparent harm. Antisera
were merthiolated 1 part to 10, 000, divided into two milliliter
amounts and frozen until used.

A modification of the slide microtechnique of Wadsworth
( 1957) was used throughout the gel diffusion studies. Two by
three inch glass slides were thoroughly cleaned in glacial acetic
acid, transferred by forceps through three washes of distilled
water and stored in absolute ethyl alcohol until needed. The
slides were air dried just prior to use.

A 0. 7 percent agar solution was prepared in 0.85 per-
cent saline. Sodium azide was added to 0. 01 molar concentra-
tion to inhibit growth of bacteria and molds. Three milliliters
of the agar solution were pipetted onto each slide. The agar
covered slides were placed in the refrigerator for five minutes

to allow the agar to gel. Holes were cut in the agar with a number

31

 

Figure 1. Gel Diffusion Slide Illustrating Configuration of

Wells for Reactants .

32

three cork borer and aspirated to form wells to contain the re-
actants . The configuration of the wells is evident by examination
of Figure 1, the central well being for the antiserum and the
surrounding six wells for the antigens.

Each of the six antisera was diffused against the six anti-
gens to determine the number of precipitin reactions taking
place between the antiserum and the homologous and heterologous
antigens.

Portions of each antiserum were absorbed by each of the
six antigens by addition of an equal volume of antigen. The
absorbed antiserum was diffused against each of the antigens.
Normal rabbit serum was diffused against all of the antigens
as a control. Precipitin lines developed by an antiserum to
heterologous antigens represent cross reactions. These pre-
cipitin lines should not appear following absorption of the
antiserum with the heterologous antigens.

In an effort to obtain antigens of excretion and secretion,
embryonated eggs of A. flan and l. aegis were hatched mechan-
ically by the method of Cleeland and Laurence ( 1962) . Approxi—
mately 100,000 embryonated eggs were washed five times with
distilled water to remove the formalin in which they had been
embryonated. The eggs were placed in a 125 milliliter Erlen-

meyer flask containing 20 milliliters of medium 199 and a one

33

inch teflon bar. The flask containing the embryonated eggs was
placed on a magnetic stirrer and run at a speed just under that
which caused frothing of the medium. The eggs of A. m were
hatched in twenty four hours. The eggs of I. 9321—5 hatched much
more quickly, four hours usually being sufficient for hatching of
almost 100 percent of the embryonated eggs.

After hatching, the larvae were centrifuged for two min-
utes at 1, 000 r.p.m. and the supernatant fluid was discarded.
The sediment containing the larvae was transferred to 20 milli-
liter screw capped tissue culture tubes which contained 7 milli-
liters of medium 199, 3 milliliters of beef serum and penicillin
and streptomycin in concentrations of 250 units each per milli-
liter of culture medium. Aseptic techniques were used through-
out the culturing procedure. Cultures were incubated at 370 C.

Gel diffusion studies were done on slides prepared as
described previously (see page 30). Freshly hatched I. _C_a_I}_i_S_
or A. m were placed in the center well and tested against
each of the five antisera used throughout this study. The antigens
of secretion and excretion of the larvae of _T_. c_ar_i_i_s_ and A. gun—m
were also diffused against the sera of mice infected for three

weeks with T. canis or A. suum. Each of five mice was infected

 

 

with 1500 embryonated eggs of A. suum. Similarly, each of five

34

mice was infected with 1500 embryonated eggs of I. Ea—nis by
stomach intubation. The sera from the five mice infected
with each species were pooled and diffused against micro-
cultures of larvae of each of the species of nematodes. More
!

media and some larvae were added periodically to the center ,
well to prevent dehydration and to aid in building up a suitable
concentration of the antigens of secretion and excretion in

the agar so reactions could occur With antibodies.

After charging the wells with reactants, in all of the
gel diffusion studies, the slides were placed in a moist
chamber which consisted of a dissecting tray with wet paper
towels on the bottom and a cover of aluminum foil. Slides
were incubated at 370 C. for forty eight hours. Reactant
wells were recharged twelve hours after incubation began.

All slides were read while still wet by means of
oblique lighting and use of a ten power hand lens. After the
first reading, slides were placed in 0. 85 percent saline for
three hours to remove some of the unreacted protein, washed
in distilled water, covered with filter paper and placed in an
incubator at 370 C. to dry. The short period of washing was

necessary as further washing obliterated some of the pre-

cipitin lines. After thorough drying, the slides were stained

35

with Ponceau S for one half hour. No de staining was done
since this made the weak precipitin lines invisible. Stained
slides could best be viewed by placing them in a photographic
enlarger where the amount of light and the magnification could
be quickly changed so all of the precipitin lines could be

seen. Hazy precipitin lines were not recorded in this study.

B. Immunoelectrophoretic studies.

Three rabbits were used in the preparation of anti-
mouse serum. Two of these rabbits were given intramuscular
injections of 0. 5 milliliter of mouse serum twice weekly for
a period of eight weeks. Following this series of injections
an intraperitoneal injection of 0. 5 milliliter of mouse serum
was given every two weeks to maintain antibody production.
Blood was removed by decapitation of mice for the first of
these injections. Later, it was discovered that blood could
be obtained from mice more quickly by intracardial puncture
with a 19 gauge hypodermic needle and syringe.

One of the two rabbits injected with mouse serum by
the above method developed a strong antibody titre to twenty
fractions of mouse serum whereas the other rabbit developed

only a weak titre to three fractions of mouse serum. Continued

36

injections of mouse serum did not stimulate further anti-
body production in this rabbit.

The third rabbit was injected by a modification of the
method of Proom ( 1943) . The following materials were
mixed.

5 milliliters of mouse serum

16 milliliters of distilled water

18 milliliters of 10 percent KAl (804); ° 12 HZO
Then 5 N NaOH was added by the drop method until no fur-
ther precipitation occurred. Following centrifugation the
sediment was washed twice with saline after which the sedi-
ment was made up to a volume of 20 milliliters in saline and
frozen until used.

The rabbit was injected according to the following
schedule. On the first and fourteenth days five milliliters
were injected intramuscularly in each buttock. On the
twenty fourth day one milliliter of untreated serum was in-
jected intraperitoneally. Thereafter, one milliliter of un-
treated serum was injected intraperitoneally every two weeks
to maintain antibody production.

Blood was withdrawn from the three rabbits used for

production of antimouse serum by intracardial puncture and

37

the serum was divided into two milliliter amounts and
frozen until used.

Two by three inch glass slides used in the immuno-
electrophoretic studies were cleaned in the same way as
those used for the gel diffusion studies (see page 30). Re-
moval of all materials which might prevent the agar gel from
adhering to the slides was necessary.

The two following buffer solutions were prepared us-
ing the method of Hirschfeld ( 1960) . The buffer to be used

in the electrode vessel contained the following materials.

Diethylbarbituric acid 13.80 grams
Sodium veronal 87. 60 grams
Calcium lactate 3. 84 grams
Distilled water to 10 1.

The buffer to be used in the agar layer was prepared as

follows.
Diethylbarbituric acid 16. 6 grams
Sodium veronal 105. 10 grams
Calcium lactate 15. 36 grams
Distilled water to 10 1.

A buffered agar gel was prepared to serve as the sup-

porting medium for the electrophoretic studies. A 1. 4 percent

38

agar. solution was prepared using ion agar produced by Con-
solidated Laboratory, ,Inc. in distilled water. Two parts of
the buffer intended for the agar gel were mixed with one part
of distilled water. The agar and diluted buffer'were heated
for five minutes in an autoclave at 20 pounds pressure after
which both were filtered. Equal parts of the diluted buffer
and the 1. 4 percent agar solutions were mixed resulting in

a 0.7 percent buffered agar solution. The agar renders the
supporting medium semi-solid and the buffer renders the
medium conductive.

Three milliliters of the buffered agar were pipetted on-
to the surface of each slide. A perfectly flat surface was
employed in order to insure even distribution of the agar
solution on the slides. The slides were then placed on 6 by 8
inch strips of plexiglass and refrigerated for five minutes to
allow the buffered agar to harden.

After the agar gel had hardened, holes were aspirated
to form wells to contain the material to be electrophoresed.
Pasteur pipettes with an inside diameter of approximately two
millimeters were selected for use in aspiration of the holes
in the agar gel. Longitudinal slits approximately three milli-
meters wide to be used to hold the antiserum following elec -

trophoresis were cut with a scalpal prior to electrophoresis

39

but they were not removed until electrOphoresis had been com-

pleted. Plexiglass templates with holes and slits cut in the

desired locations were used to process the slides more quickly.
Albino mice of the Rockland strain, each weighing

25-30 grams, were infected with 1500 embryonated eggs of

T. canis or A. suum. The embryonated eggs were washed

five times in distilled water to remove the formal in which

embryonation had occurred. Five counts of 0. l milliliter

each were taken of a suspension of embryonated eggs.

The average of these five counts was used as a basis for in-

fection of the mice. Mice were lightly etherized and the eggs

were introduced by stomach intubation with a Pasteur pipette.

Each of five mice was infected with 1500 embryonated eggs

of T. canis. The same procedure was used in infecting each

 

of five mice with 1500 embryonated eggs of A. m.
Samples of blood were drawn from the mice prior to

infection and every three days thereafter for a period of three

weeks following which the infected mice were exsanguinated

and the serum was frozen for future use. Blood was with-

drawn from each of the infected mice by clipping off the tip

of the tail and filling heparinized capillary tubes with blood.

One end of the tube was plugged with clay. The tubes were

40

centrifuged for five minutes at 4000 r.p.m. . The portion of
the capillary tube containing the serum was broken off and the
serum was transferred to a tuberculin syringe using a 27 gauge
needle. A small drop of this serum was placed in a hole
aspirated from the agar covered slide just prior to electro-
phoresis.

The slides were prepared so that the sera of mice in-
fected with A. m, normal mice and mice infected with I.

canis could be directly compared on the same slide. The con-

 

figuration of the slides can be seen in Figure 4.

After the serum from the mice infected with A.fl1_1_1r_n,
normal mice and mice infected with I. 2% was placed in
the wells, a constant current of 110 volts was passed through
the buffered agar gel for one and one -half hours . A voltage
meter was used to test the actual voltage through the buffered
agar on the slides.

The longitudinal slits which had been cut in the agar
before electrophoresis were now removed by aspiration to
form troughs. These troughs were filled with antimouse serum
which had been developed in rabbits. Slides were then placed
in a moist chamber consisting of a covered dissecting tray

0
containing some water and were incubated at 37 C. for twenty

41

four hours to allow development of precipitin lines.

In order to observe more clearly the precipitin lines
which seemed to represent increases in amounts of certain
globulin fractions during the course of infection, dilutions
of the sera of infected mice were run by electrophoresis and
precipitin lines were developed by undiluted antimouse serum.
The diluent was 0. 85 percent saline which did not react with
antimouse serum when run as a control.

Five mice were infected with 1500 embryonated eggs

of A. suum and five mice were infected with 1500 embry-

 

onated eggs of I. 2321—5 by the method previously described
(see page 39) . The mice were bled by a tail vein and 1 to 16
and 1 to 32 dilutions of the sera were electrophoresed previous
to infection and every week for a period of three weeks fol-
lowing infection.

Infectivity of the eggs was checked by autopsy of a
mouse presumably infected with each of the roundworms seven
days after the eggs were given to them. Larvae of each of the
species were observed.

In an effort to determine if changes in the globulin frac-
tions of the serum during the course of infection were due to

antibody production, sera from mice with a three week infection

42

with these roundworms were absorbed with antigens prepared
from the various stages in the life cycles of the roundworms.
The antigens used in these absorption studies were prepared
as described above for the gel diffusion studies (see page 28) .
Antigens of unembryonated eggs, embryonated eggs and whole
worms of each of the species were used to absorb serum
specimens from mice infected with fix-11132: uninfected con-

trol mice and mice infected with I. canis. These three serum

 

specimens, absorbed by an equal amount of one of the anti-
gens, were electrophoresed on the same slide and precipitin
lines were developed by diffusion against full strength anti-
mouse serum. Comparisons could then be readily made.
The antigens were run by electrophoresis as controls and

they did not react with antimouse serum.

RESULTS

A. Gel diffusion studies.

A series of two-fold dilutions of the antigens in 0.85
percent saline was diffused against homologous antisera to
determine the dilution of each antigen which produced the
maximum number of precipitin lines . One to two dilutions
of the antigens of adult whole worms of A.fl_1_1_r_n and I. 93313
produced the maximum number of precipitin lines when the
antigens were diffused against homologous antisera. One to
four dilutions of the remaining antigens produced the maximum
number of precipitin lines when diffused against their homol-
ogous antisera. Dilutions of the antigens producing the
greatest number of precipitin lines were used throughout the
gel diffusion studies.

The antiserum to adult whole worms of A. su_u__m was very
weak, producing only four distinct precipitin lines with its
homologous antigen. The antiserum to adult whole worms of

I. canis was strong, producing eight precipitin lines with the

43

44

homologous antigen. Since the antiserum to adult whole

worms of T. canis would disclose all of the cross reactions

 

to the antigen from adult whole worms of A. w, only the
former antiserum was used throughout this study. No pre-
cipitin lines developed when normal rabbit serum was dif-
fused against all of the antigens as a control. The following
numbers of precipitin lines were developed when the remaining
antigens were diffused against their homologous antisera: six
with the unembryonated egg antigen of A. w, six with the
unembryonated egg antigen of I. c_ar§_s_, four with the embry-
onated egg antigen of A. su____1i__m and four with the embryonated

egg antigen of _T_. canis.

 

Precipitin lines developed by an antiserum to heter-
ologous antigens represent cross reactions. When the anti-

serum to adult whole worms of _T_. canis was used to develop

 

precipitin lines with the five heterologous antigens, the follow-
ing numbers of precipitin lines were observed: eight with the
antigen of adult whole worms of A. m, five with the anti-
gen of unembryonated eggs of I. gags, one with the antigen of
unembryonated eggs of A. M and none with the antigens of
the embryonated eggs of either species.

Table 1 summarizes the numbers of precipitin lines

developed when the antigens to different stages of the life cycles

45

of the two species of roundworms are diffused against
homologous and heterologous antisera.

If the antigen from the adult whole worms of I. canis

 

was diffused against the heterologous antisera, a corres-
ponding number of precipitin lines should appear as in the
reciprocal procedure described above. Table 1 shows the
following numbers of precipitin lines developed when the
antigen of adult whole worms of I. SEE was diffused against
the various heterologous antisera: five with the antiserum to
the unembryonated eggs of _T. 03-1—8, one with the antiserum
to the unembryonated eggs of A. s_m_1_r_n, none with the anti-
serum to the embryonated eggs of A. m and three with the
antiserum to the embryonated eggs of I._C_al_'1_i_S. When the anti-
serum to embryonated eggs of _'I_‘. Elli—S was diffused against

antigen of adult T. canis, three precipitin lines formed where-

 

as the reciprocal procedure produced no precipitin lines.
Precipitin lines demonstrated by diffusion of an antiserum

against the various heterologous antigens should not appear fol-

lowing absorption of the antiserum with the heterologous antigens.

When the antiserum to adult whole worms of I. canis was ab-

 

sorbed by the heterologous antigens and subsequently diffused

against these antigens, none of the precipitin lines representing

46

Table 1. Numbers of precipitin lines developed when anti-
sera are diffused against homologous and heterologous

antigens .

 

Antigens
Antiserum A B C D E
A 8 8 5 1 0
C 5 2 6 2 1
D 1 4 2 6 0
E 3 1 4 0 4
F 0 3 0 3 0

 

A - adult whole worms of I. canis

 

B - adult whole worms of A. suum

 

C - unembryonated eggs of I. canis

D - unembryonated eggs of A. suum

 

E - embryonated eggs of _T_‘. canis

F - embryonated eggs of A. suum

47

cross reactions to these antigens developed as shown in
Table 2.

Comparison of the data in Tables 1 and 2 reveals the
number of precipitin lines which developed when the anti-

gen from adult whole worms of I. canis was diffused against

 

its homologous antiserum as compared to the number of
precipitin lines which developed following absorption of the
antiserum with the heterologous antigens. Absorption of

the antiserum with its homologous antigen acted as a control.
The number of precipitin lines removed by absorption of the
antiserum with heterologous antigens and subsequent develop-
ment of the absorbed antiserum with the heterologous antigens
represents the number of cross reactions.

If the reactions of the antigens with each of the anti-
sera are analyzed in the same way as was done for the anti-
serum to adult whole worms of _T. 2312, several unexpected
results are revealed. Examination of Table 1 shows that when
the antigen of unembryonated eggs of 2.211}: was diffused
against antiserum to embryonated eggs of the same species
four precipitin lines developed whereas only one developed
with the reciprocal procedure. Also, when the antiserum to

the unembryonated eggs of A. suum was diffused against the

48

adult whole worms of I.

adult whole worms of A.

unembryonated eggs of I.

unembryonated eggs of A.

embryonated eggs of _T_.

embryonated eggs of A.

canis

 

S uum,

 

canis

 

S uum

 

canis

 

S uum

 

49

Table 2. Numbers of precipitin lines detected when antigens

are diffused against antisera A and C absorbed with each

 

 

.- antigen.
Antiserum plus Antigens
absorbing antigen A B C D E
A + A 0 0 0 0 0
A + B 2 0 0 0 0
A + C 2 2 0 0 0
A + D 5 2 1 0 0
A + E 3 3 1 0 0
A + F 4 2 2 1 0
C + A 0 2 0 O 0
C + B 2 0 3 0 0
C + C 0 0 0 0 0
C + D 3 0 l 0 0
C + E 4 2 5 0 0

 

50

embryonated eggs of the same species two precipitin lines
were observed but with the reciprocal procedure three pre-
cipitin lines appeared.
The numbers of precipitin lines developed after ab-
sorption of the antiserum to the unembryonated eggs of I.
3% and A.fl1_1_1_r_n by the various antigens are shown in Table 2
and Table 3 respectively. The data in relation to the antiserum
to the embryonated eggs of A.§1_11_Jin are presented in Table 4.
Five precipitin lines developed when the antiserum

to adult whole worms of _T_. canis was diffused against the

 

antigen of unembryonated eggs of the same species. The
reciprocal procedure also produced five precipitin lines.
These five precipitin lines represented cross reactions. An
examination of double diffusion slides demonstrated the iden-

tity of three antigens in the adult whole worms of I. canis with

 

three antigens in the unembryonated eggs of the homologous
species. Reciprocal absorption of the antisera with antigens
indicated that there were cross reactions other than the reac-
tions of identity.

Three reactions of identity developed between antigens

of whole worms of T_. canis and embryonated eggs of the same

 

species when these antigens were diffused against the antiserum

to embryonated eggs of _T_. canis.

51

adult whole worms of I. canis

adult whole worms of A. suum

 

unembryonated eggs of I. canis
unembryonated eggs of A. suum
embryonated eggs of _T_. canis

embryonated eggs of A. suum

 

52

Table 3. Numbers of precipitin lines observed when antigens

are diffused against antisera D and E absorbed with each

 

antigen.
Antiserum plus Antigens
absorbing antigen A B C D E
D + A 0 4 0 3 0
D + B 0 0 0 l 0
D + C 0 3 0 3 0
D + D 0 0 0 0 0
D + E 0 3 l 3 0
D + F 0 4 2 4 0
E + A 0 0 0 0 2
E + B 0 0 0 0 2
E + C 0 0 0 0 2
E + D 2 0 3 0 2
E + E 0 0 0 0 0

 

53

Table 4. Numbers of precipitin lines demonstrated by dif-

fusion of antigens against antiserum F absorbed with each

 

antigen.
=1
Antiserum plus Antigens
absorbing antigen A B C D E
F + A 0 2 0 2 0
F + B 0 0 0 0 0
F + C 0 0 0 0 0
F + D 0 0 0 0 0
F + E 0 2 0 2 0
F + F 0 0 0 0 0

 

 

 

A - adult whole worms of _T_. canis
B - adult whole worms of A. suum
C - unembryonated eggs of I. 53313
D - unembryonated eggs of A. m

E - embryonated eggs of _'I_‘. canis

F - embryonated eggs of A. suum

 

54

Only one reaction of identity was observed among the
antigens of the unembryonated eggs, embryonated eggs and

adult whole worms of _T_. canis. The reaction of identity was

 

demonstrated by diffusion of the above antigens against the
antiserum to the unembryonated eggs of _T_. m. Absorp-
tion studies showed that there were cross reactions other
than those revealed by reactions of identity.

Two precipitin lines developed when the antiserum to
unembryonated eggs of A. m was diffused against the anti-
gen from unembryonated eggs of I. gag. The reciprocal
procedure yielded two precipitin lines as shown in Table 1.
These two precipitin lines represented cross reactions. In
order to determine if these were identical antigens, double
diffusion slides were examined for reactions of identity.
There were two reactions of identity when the antiserum to
unembryonated eggs of A. m was diffused against the anti-

gens of unembryonated eggs of A. suum and I. canis. The

 

reciprocal procedure also resulted in two reactions of identity.
The two cross reacting antigens were removed by absorption
of the antiserum to unembryonated eggs of A. _s_u_1_1_r_r_1 with the
antigen to unembryonated eggs of I. SE13 as shown in Table 3,

The results of mutual absorption studies of the antisera show

55

that there were reactions other than those revealed by the
reactions of identity.

When microcultures of living larvae were diffused against
the antisera used throughout this study, one precipitin line
developed with the antiserum to embryonated eggs of the
homologous species. No cross reaction was observed.

When microcultures of living larvae were diffused
against the sera of mice infected with each of the species
of nematodes no clearly defined precipitin lines were dis-
cernible A hazy area appeared in the agar when larvae of
each species was diffused against the sera of mice infected

with that species indicating that precipitins were present.

B. Immunoelectrophoretic studies.

When normal mouse serum was run by electrophoresis
and precipitin lines were developed with antimouse serum,
twenty clearly distinguishable precipitin lines developed. Not
all of these precipitin lines were visible on the same slide. From
the most anodic to the most cathodic in position, these precipitin
lines represent the following serum fractions: 1 albumin,
2 alpha -one globulins, 8 alpha-two globulins, l beta-one globu-
lin, 4 beta-two globulins, 3 beta-three globulins and 1 gamma

globulin. The relative positions of these precipitin lines are

56

shown in Figure 2. The precipitin lines were named accord-
ing to the nomenclature of Heremans it Ei' ( 1959) who worked
out the immunoelectrophoretic characteristics of normal mouse
serum.

Heremans e_t a_l. (1959) observed precipitin lines repre-
senting a pre-albumin and 2 alpha -one globulins which were
never observed during the course of this study. However, this
study revealed two alpha -two globulins that were not recorded
by the previous investigators. If one draws a perpendicular
from the widest point of the precipitin arc to the path of electro-
phoretic migration, the center of concentration of the antigen
responsible for the develOpment of the precipitin line can be
located. The centers of concentration for two of the antigens
called alpha-twos by Heremans St 31. (1959) lie much farther
anodically than do the centers of concentration for the remaining
antigens called alpha -twos. In the present study the centers of
concentration for six of the alpha antigens lie close together
and much farther cathodically than the remaining two alpha
antigens as seen in Figure 3.

The immunoelectrophoretic pattern of normal mouse
serum as deveIOped by each antimouse serum must be ascertained

before the antimouse serum can be used to determine the effects

57

Figure 2. Composite of Immunoelectrophoretic Patterns

of Normal Mouse Serum.

\n
0‘)

:21 l

L——-————-— ,

a, 111
5311

F331

{3211

5,111

 

 

 

[521V

 

) ._—-—— antigen well

_______ _ antiserum trough

 

 

____________ 5,1
________-_ QVL QVH,aflHH

_________ ca 111, a2 IV, aZV

______ _. (12 II

——————— all

alII
-_______._——— all

______________ albumin

 

 

 

Figure 2.

59

521

fill

az VI, VII, V111

«12111, 1v, v

all].
(121

albumin

 

Figure 3. Alpha-two Components in Normal Mouse Serum.

60

of parasitic infection on the serum proteins of mice. After
the electrophoretic patterns of normal mouse serum had
been studied, an effort was made to secure comparative data
on the effects of infection with I. 2111—5: and A. _s__1_111_r_n on the
serum of individual mice . Five mice were infected with
1500 embryonated eggs of I. 551—111: and five mice were in.-
fected with 1500 embryonated eggs of A §_1_1_1_J_r_r_1. A series of
slides was prepared every three days for three weeks. Al-
though three series of slides were prepared in an effort to
secure comparative data on the effects of infection on indi-
vidual mice, the results of immunoelectr0phoresis were found
to be too inconsistent to justify such comparisons. However,
the large number of slides accumulated as rthe result of this
effort became very useful in establishing changes which oc-
curred in the serum globulins of mice during the course of
infection with these two species of roundworms.

An apparent increase in the a 211 serum globulin of
mice infected with A. m was revealed by comparison of
the electrophoretic pattern of serum from a normal mouse with

that of a mouse infected for eighteen days with A. suum as

 

shown in Figure 4. In order to determine whether this was

an apparent or real increase in the amount of this globulin

61

 

Figure 4. Immunoelectrophoretic Patterns of Undiluted
Mouse Serum: from left to right-~A. suum

infected, normal, I. canis infected.

62

fraction, serum dilutions of 1 to 16 and 1 to 32 were prepared
each week for a period of three weeks from five mice infected

with A. suum. These serum dilutions were run by electro-

 

phoresis and developed by undiluted antimouse serum. By com-
parison of the diluted serum from the infected mice with the
serum from the uninfected control mice which was similarly
diluted, the increase in the (2211 serum globulin became evident
on the seventh day following infection as shown in Figure 5.
There was an increase in the amount of gamma globulin

in mice infected with I. canis as shown in Figure 5. This

 

increase became apparent on the seventh day following in-
fection and persisted through the third week of infection as
revealed by serum dilutions which were electrophoresed and
diffused against full strength antimouse serum.

Dilution of the serum prior to electrophoresis proved
to be a very satisfactory method of detecting increased
amounts of various serum components. Without dilution the
increased components were often masked by other components.

The sera of infected mice were absorbed with antigens
in order to determine if the alterations in the serum globulin
fractions during infection were due to antibody production.

The pooled sera of five mice infected for three weeks with

63

 

Figure 5. Immunoelectrophoretic Patterns of Diluted Mouse
Serum: from left to right --A. suum infected,

normal, T. canis infected.

64

A. £32211, uninfected control mice and five mice infected for
three weeks with I. ga_n_i_s were absorbed with the antigens pre-
pared from unembryonated eggs, embryonated eggs and adult
whole worms of A. w and I. C_an_i_s_. This procedure would
reveal if antigens in any of these stages of the life cycles of
these roundworms would absorb the material in the increased
fractions of globulin in the infected mice. Following electro-
phoresis of the absorbed sera they were diffused against full
strength antimouse serum.

Neither the a 211 fraction in the serum of mice infected
with A. w nor the gamma globulin in the serum of mice

infected with I. canis was affected by absorption of sera with

 

the various antigens. Figure 6 shows the results of absorption

of the serum of mice infected with A. suum with the antigen

 

prepared from the adult whole worm of A. suum. Figure 7
shows the results of absorption of the serum of mice infected

with I. canis with the antigen prepared from the adult whole

 

worms of T. canis.

 

65

7* 'azII

 

Figure 6. Immunoelectrophoretic Patterns of Mouse Serum:
from left to right—-normal, absorbed with antigen
of adult A. _sluLn; A. w infected, absorbed
with antigen of adult A. m; A minfected,

unabsorbed.

66

 

Figure 7.

Immunoelectrophoretic Patterns of Mouse Serum: from
left to right "I. canis infected, unabsorbed; 'I_‘. canis
infected, absorbed with antigen of adult I. canis;

normal, absorbed with antigen of adult T. canis.

DISCUSSION

A. Gel diffusion studies .

When the antiserum to the adult whole worms of I. canis

 

was diffused against its homologous antigen, eight precipitin
lines developed. Two precipitin lines developed when the
antiserum was absorbed with the antigen of adult whole worms
of A. §u_u_n_i and diffused against the antigen of adult I. ga_n_i_s_.

No precipitin lines appeared when the absorbed antiserum was

 

diffused against antigen of adult A. suum. If the eight anti-

bodies which cross reacted with A. suum are removed from the

 

antiserum by absorption and two still remain to react with anti-

gens of adult I. canis, there must be a greater complexity of

 

antigens and antibodies present than was revealed by diffusion
of the antiserum against its homologous antigen.
The two precipitin lines which developed when the anti-

serum to whole worms of T. canis was absorbed by whole worm

 

antigen of A. suum and diffused against antigen of whole worms

 

of T. canis represent specific antigens of adults of I. canis when

67

68

compared to the same stage of development in the life cycle
of A.§1_1_Lir_n. Even though these are specific antigens present
in the adult stage of I. 23%, they are probably not functional
antigens since there is considerable evidence that the larval
stages rather than the adult. stages of para51ti.c nematodes
elaborate the functional antigens.

As previously stated, eight. precipitin lines developed

when full strength antiserum to whole worms of T. canis

 

was diffused against the homologous antigen. Eight prel-
cipitin lines were also observed when this antiserum was

diffused against the antigen of whole worms of A. suum as

 

shown in Table 1, indicating eight. cross reactions.
Kagan (1957), using the reciprocal procedure, found

that full strength antiserum to A. suum adults developed ten

 

precipitin lines when diffused against the homologous antigen.
Four precipitin lines appeared when the antiserum to adult A.

suum was diffused against the antigen to whole worms of I.

 

canis. If the antiserum to A. suum adults produced ten pre-

 

cipitin lines with homologous antigen and four with the antigen

of adult T. canis then six specific antigens are indicated in

 

the adult stage of A. suum when compared to the adult stage of

 

T . canis .

69

If there are six specific antigenic components present

in the antigen of whole worms of A. suum as indicated by the

 

work of Kagan ( 1957) and in addition eight antigenic components
which cross react with the antiserum to whole worms of T.
canis as indicated in the present study, then at least fourteen

antigenic components must exist in the whole worms of A. suum.

 

Kagan ( 1957) concluded by different reasoning that a minimum of
fourteen antigenic components were present in the antigen of

whole worms of A. suum.

 

Full strength antisera were used in all absorption studies
during this investigation whereas Kagan ( 1957) used a single
antiserum diluted one to four. Comparisons are therefore
difficult.

In the present study when the antiserum to whole worms

. of T. canis was absorbed with antigen of unembryonated eggs

 

of A. suum and diffused against the antigen of whole worms of

 

A. suum, two precipitin lines were observed whereas this

absorbed antiserum developed five precipitin lines when

diffused against antigen of whole worms of I. canis . This

 

indicated that there were three antigens present in the un-
embryonated eggs of A. suum which cross reacted with anti-

bodies in the antiserum to whole worms of T. canis.

70

The work of Kagan ( 1957) indicated that the unembry-
onated eggs of A. 323111 contained all the antigens present in
the adult stage of this species. In the present study absorp-
tion of antiserum to unembryonated eggs of. I. 32333 with the
antigen of whole worms of the same species indicated the
adult worms contained all antigens present in the eggs. The
reciprocal procedure demonstrated that the unembryonated
eggs did not contain all of the antigens in the adult worm.

The development of precipitin lines when the antiserum
to embryonated eggs of I. 33311.2 was diffused against antigen
of the adult worms of the same species and the lack of‘develop-
ment of precipitin lines with the reciprocal procedure may be
due to the great differences that exist in the ability of rabbits
to produce antibodies. Another possible explanation is that
the two antigens used were not of equal potency.

Hyperimmune animals develop antibodies which display
great cross reactivity (Ouchterlony 1962) . The antiserum to
the embryonated eggs of _T_. M used in this study maybe
from a hyperimmune rabbit. since great cross reactivity was
displayed by this antiserum.

The development of precipitin lines when microcultures

of living larvae of I. canis and A. Vs_uum were diffused

 

71

against the antiserum of embryonated eggs of the homologous
species is not surprising since the larvae were in the same
stage of development as they were in the embryonated eggs.

Lack of development of clearly defined precipitin lines
when microcultures of living larvae of each species were dif-
fused against the serum of mice infected with these species
may be due to the unavoidable bacterial contamination in the
well containing the larvae .

The antigens in the two nematodes studied are potent
allergins. After almost daily exposure to these antigens
for eighteen months, a differential white blood count of the
present investigator revealed an eosinophilia of 45 percent.
Skin contact resulted in an immediate hypersensitive response
of edema and a delayed response of dermatitis. Inhalation
resulted in symptoms of hayfever and asthma. Serum tested
by double diffusion against all of the antigens used in the pres-
ent study revealed no precipitins. Sprent ( 1949) found that

the antigens of A. suum caused skin sensitivity in humans

 

without the development of precipitins.

B. Immunoelectrophoretic studies.

Immunoelectrophoresis brings about resolution of the

72

globulins into many individual components where minute
qualitative and structural abnormalities as well as quan-
titative changes of each component may be studied.

The increase in the a zII globulin fraction in mice infected
with A.§1_11;n may be due to inflammation or tissue damage.
Shedlovsky and Scudder ( 1942) found that there was an increase
in alpha globulin under these conditions regardless of their
cause.

The liver has been indicated as the site of production
of the alpha and beta globulins (Leland 1961) . Peters and
Anfinsen ( 1950) found that the liver was also the site of
production of the serum albumin. Sprent ( 1952) found that

in mice infected with A. suum the larvae remained in the

 

 

liver for four days whereas in mice infected with _T_. canis
the larvae left the liver much sooner. Perhaps the presence
of A. m larvae in the liver for this extended period of time
results in considerable tissue damage and inflammation which
effects an increase in the rate of synthesis of (1211 fraction of
the serum.

The possibility that there was a decrease in albumin dur-
ing infection was considered although the immunoelectrophoretic

technique would not reveal such a reduction. Leland ( 1961)

73

stated that osmotic pressure was maintained largely through
the albumin fraction so that any loss of albumin might result
in a compensatory reaction to restore serum osmotic pressure.
Miller and Bale ( 1954) found evidence that the alpha globulins
are produced and turned over at a more rapid rate than other
serum protein fractions. Therefore, the alpha globulins
might very well take over the function of lost albumin.

Plasma proteins may be lost in severely affected animals
through the damaged mucosa of the alimentary tract. A loss

of plasma proteins in this way in mice infected with A. suum

 

could result in synthesis of increased amounts of alpha globulin
to compensate for this loss.

The a 211 globulin in mouse serum was shown to be related
to human haptoglobin by Clausen and Heremans ( 1960) . This
was done by the immunoelectrophoretic method using a specific
antihaptoglobin antiserum obtained by complete absorption of an
antihuman antiserum with the serum from a patient with per-
nicious anemia. In this disease there is a startling decrease
in circulating serum haptoglobin. The antihaptoglobin antiserum
developed only one precipitin line with human serum and only one
line with mouse serum which corresponded to the a all fraction

of mouse serum.

74

The discovery that the increase in the a all fraction in

the serum of mice infected with A. suum is not due to anti-

 

body production to the antigens tested is not surprising
since most antibodies are known to be associated with the
gamma globulin in the serum. That, the increase in gamma
globulin in the serum of mice infected with I. (3% is ap-
parently not due to antibody production, as demonstrated by
the absorption studies performed, is somewhat: surprising.
However, these tests show only that if there are antibodies

present to A. suum or I, canis antigens, that these anti-

 

bodies were not produced by the mice as a result of exposure
to the antigens used for absorption. Excretion and secretion

of antigens by the larvae of A. suum or _T_. canis in the in—

 

 

fected mice could cause antibody production. These anti-
bodies might not be removed by absorption of sera with the
antigens used.

The increased amount of gamma globulin in the serum
of mice infected with I. 23111:; may be due to secondary bac-
terial invasion following larval penetration of the intestine.
This is a rather remote probability since the larvae of A.
m also penetrate the intestine and no increase in the

amount of gamma globulin is observed in the serum of mice

75

infected with this roundworm. However, the newly hatched

larvae of I. canis were observed to be much larger than

 

those of A. suum. The larvae of I. canis may damage the

 

tissues of the intestine to a greater extent than those of A.
mthus paving the way for secondary bacterial invasion.
The possibility that the increase in gamma globulin
was only relative to the other serum proteins due to main-
tenance of the gamma globulin level while other fractions
decreased in amount was considered. Comparison of di-
luted serum of uninfected mice with the diluted serum of

mice infected with T. canis, as shown in Figure 7 revealed

 

that there was a real increase in amount of gamma globulin

in mice infected with T. canis.

 

The six antigens used in gel diffusion studies were
electrophoresed in an attempt: to secure precipitin lines by
development of the electrophoresed antigens by serum from

mice infected with A. suum or I. canis. Although this was

 

 

done every three days for a period of three weeks after in-
fection, no precipitin lines were observed which indicated
lack of development of precipitating antibodies by the mice
or that if precipitating antibodies were produced they never
reached equivalence with antigens in this experimental

procedure.

76

Kent (1960) found that glycoprotein complexes isolated
from the water soluble portion of adult A. s_uu_m could be
separated into five major components by agar gel electro-
phoresis. Four we re active immunologically and they seemed
to be somewhat specific since sera from suspected cases of
visceral larva migrans, presumably of 2383:5133 origin, did
not react with the antigens of two human patients twelve days
before the eggs were found in the stools.

Huntley and Moreland ( 1963) demonstrated precipitins
to water soluble antigens of adult whole worms of I. c_a_£1_i_s in
two children with visceral larva migrans, however, one of
these children also had precipitins which reacted with the

water soluble antigens of A. lumbricoides indicating a cross

 

reaction. Cross reactions between closely related ascarids
have been noted by Kagan, e_t fl. ( 1959), using the bentonite
flocculation test; Olson ( 1960), using the i_n Ltrg larval
precipitate test; Huntley and Moreland (1963), using gel
diffusion techniques .

There is evidence that a decrease in serum albumin
and an increase in globulin components of the serum occurs
in animals infected with certain parasites (Stauber 1954:) .

Many parasitic infections cause an anemia in the host. Changes

77

in serum proteins in animals infected with parasites could be
directly due to the anemia and not to the parasite. Kuttler
and Marble ( 1960) found that in producing an experimental
anemia by bleeding sheep and comparing their serum proteins
to those of heavily parasitized sheep that the heavily parasitized
sheep had a relatively lower albumin and higher alpha -two
globulin fraction than the sheep with the induced anemia.

The changes noted in the present study in the globulin

fractions of mice infected with A. suum and I. canis are not

 

 

specific to the infections.

SUMMARY AND C ONC LUSlONS

A. Summary.

A comparative antigenic analysis of the unembryonated
eggs, embryonated eggs and adults of I. C_a;r_1_i_s and A. w
was completed by the double diffusion slide microtechnique
of Wadsworth ( 1957). Diffusion of antisera to adult whole
worms of I. canis, unembryonated eggs and embryonated
eggs of I. 23:5 and A. suum against homologous antigens
revealed the number of antigenic components in each of the
antigens. Cross reactions were demonstrated by diffusion
of each antiserum against heterologous antigens.

Diffusion of the sera of mice infected with_A_. w
or 2.93113 against the antigens of excretion and secretion
from microcultures of living larvae of these roundworms
demonstrated the presence of weak precipitins in the serum
of the infected mice. Further research is indicated to clarify
the role of antigens of excretion and secretion from larval

stages of A. suum and I. canis in the development of anti-

 

 

bodies which provide immunity to subsequent infection.

78

79

The effects of infection with I. canis and A. suum on

 

 

the serum proteins of mice were investigated by immuno-
electr0phoresis. There was an increase in call serum globu-
lin in mice infected with A. m. The serum of mice infected
with _T_. EELS showed an increase in gamma globulin. These
alterations in serum globulin fractions during infection are
not due to production of antibodies to the antigens found in the
unembryonated eggs, embryonated eggs or adult stages of the
species with which the mice were infected. Absorption of the
sera of infected mice with these antigens had no effect on the
increased globulin fractions.

The increase in the amount of a all serum globulin in

mice infected with A. suum was not specific to the roundworm

 

but was apparently the result of inflammation and tissue damage,

particularly in the liver. The larvae of A. suum remain in the

 

liver much longer than those of T. canis. The increase in

 

gamma globulin in mice infected with I. canis was indicated
to be non-specific to the helminth. Secondary invasion of
the tissues of the intestine by bacteria following entrance of

the larvae of T. canis could account for an elevation in the

 

amount of gamma globulin. Since the larvae of _'l_‘. canis are

 

much larger than those of A. suum their penetration of the

intestine would effect easier access by bacteria.

80

The results of diffusion of antigens of excretion and
secretion from microcultures of living larvae against anti-
sera to embryonated eggs and sera of infected mice revealed
no cross reactions between species. Cross reactions were
indicated in all other experimental procedures. Because of
this, further research is indicated to determine if the embry-
onated eggs or cultures of larvae could be used as a source of

antigen in the serological diagnosis of visceral larva migrans .

B. Conclusions.
1. Diffusion of antigens against homologous antisera
indicated the following numbers of antigenic components in

various stages of the life cycles of T. canis and A. suum:

 

six in the unembryonated eggs of both species, four in the
embryonated eggs of both species, eight in the adult stage
of I. 339:8. No antiserum to adult 5 _s_uurli was used in this
study.

2. Cross reactions were demonstrated by diffusion
of each antiserum against the heterologous antigens . The
results indicated that most antigenic components cross reacted
with the heterologous species.

3. Diffusion of the antigens of adult worms of A. suum

 

against the antiserum to adult _'I_‘. canis demonstrated eight

 

81

cross reacting antigenic components in the adults of A. suum.

 

4. Absorption of the antiserum to adult 2.25.135: with the
antigen to adult A. £3921 and diffusion of the absorbed anti-
serum against the antigen of adult I. 23135 produced two
precipitin lines. These two precipitin lines represent specific

antigens in the adult stage of "E. canis when compared to the

 

adult stage of A. suum.

 

5. Lack of mutual absorption of antisera with antigens
indicated a greater complexity of antigenic components
in unembryonated eggs, embryonated eggs and adult worms

of A. suum and T_. canis than was revealed by diffusion of

 

 

antisera to each of the stages in the life cycles of these
roundworms against their homologous antigens.
6. Antigens of excretion and secretion from micro-

cultures of newly hatched larvae of _T. canis and A. suum

 

 

developed precipitin lines when diffused against antisera to
embryonated eggs of the homologous species.
7. Precipitins in the serum of mice infected with A.

suum or I. canis were indicated by hazy areas of precipitation

 

in the agar when the serum of infected mice was diffused
against microcultures of living larvae of the species with which

the mice were infected.

82

8. Normal mouse serum contained twenty fractions
as revealed by the technique of immunoelectr0phoresis.

9. The alpha -two fraction of mouse serum was
found to contain two components not reported by previous
investigators .

10. Antimouse sera produced in rabbits varied tre-
mendously in the number of fractions to which antibodies
were present and in the amount of antibody present to the
various components of mouse serum.

11. There was an apparent increase in the call fraction
of the serum of mice infected for one week with 1500 larvae

of A. suum. Electrophoretic patterns of dilutions of serum

 

from the infected mice proved that the apparent increase in
the call fraction was not due to maintenance of the call level
while other serum components decreased in amount.

12. The increase in amount of the azll in the serum of
mice infected with A. glint—n was not due to production of anti-
bodies to antigens in the unembryonated eggs, embryonated

eggs or adult worms of A. suum as indicated by the absence

 

of absorption of the increased fraction of serum with these
antigens .

13. The increase in amount of the call fraction of the

83

serum of mice infected with A. m was not specific to the
roundworm.

14. An increase in the amount of gamma globulin in the
serum of mice infected with 1500 larvae of I. gigs became
apparent one week following infection.

15. Electrophoretic patterns of dilutions of the serum
of infected mice revealed that there was an actual increase in
amount of gamma globulin rather than a maintenance of the
level of gamma globulin while other components decreased
in amount.

16. The increase in amount of gamma globulin in mice

infected with I. canis was not due to production of antibodies

 

to antigens in the unembryonated eggs, embryonated eggs or
the adult of I. Lnis because the serum of infected mice was
not affected by absorption with these antigens .

17. The change in the serum globulin of mice infected

with _T_. canis was apparently not specific to the helminth.

BIBLIOGRAPHY

Abramson, Harold Alexander. 1934. Electrokinetic Phe-
nomena and Their Application to Biology and Medicine.
New York: The Chemical Catalog Company, Inc. 331 pp.

Andersen, F. L., D. J. Graff, D. M. Hammond, P. R. Fitz-
gerald, and M. L. Miner. 1960. Changes in the blood

of calves experimentally infected with Ostertagi_a oster-

 

tagi or Haemonchus placei. J. Parasit., 46(Section 2):

 

115, 116.

Atchley, F. 0., A. H. Auerheimer, and M. A. Wesley. 1961.
Immunoelectrophoresis on blood serum proteins of guinea
pigs with experimental amebiasis. J. Parasit., 48:
229-232.

Beaver, P. C., C. H. Snyder, G. M. Carerra, S. H. Dent,
and J. W. Lafferty. 1952. Chronic eosinophilia due to
visceral larva migrans. Pediatrics, 917-19.

Beaver, P. C. 1956. Larva migrans. Exp. Parasitol...

5:587-621.

84

85

Bier, Milan. 1959. Electrophoresis. New York: Academic
Press, 563 pp.
Campbell, D. H. 1936. An antigenic polysaccharide fraction

of Ascaris lumbricoides. J. Inf. Dis., 59:266-280.

 

Campbell, D. H. 1937. The immunological specificity of
a polysaccharide fraction from some common parasitic
helminths. J. Parasit., 23:348-353.

Campbell, Charles M. 1955. The antigenic role of excretions

and secretions of Trichinella spiralis in production of

 

immunity in mice. J. Parasit., 41:483—491.
Canning, G. A. 1929. Precipitin reactions with various tis-

sues of Ascaris lumbricoides and related helminths.

 

Amer. J. Hyg., 9:207-266.

Clausen, J., J. Heremans, M. Th. Heremans, and R. Rask-
Nielsen. 1959. Immunoelectrophoretic studies of serums
from mice carrying two transplantable plasma cell leu-
kemias. J. Nat. Cancer Inst... ZZ:57~81.

Clausen, J., and J. Heremans. 1960. An immunologic and
chemical study of the similarities between mouse and
human serum proteins. J. Immunol., 84:128-134.

Cleeland, Roy, and Kenneth A_ Laurence. 1962, liivitro

 

cultivation of Ascaris lumbricoides variety suum

 

larvae. J. Parasit., 48:35-38.

86

Cleeland, Roy. 1963. The effects of different atmospheres
and various supplements on i_n vitro survival and growth

of Ascaris suum larvae. J. Parasit., 49:64-68.

 

Crandall, Catherine A., Rene Echevarria, and Victor M.
Arean. 1963. Localization of antibody binding sites

in the larvae of Ascaris lumbricoides variety suum by

 

means of fluorescent techniques. Exp. Parasitol.,
14:296-303.

Crowle, Alfred J. 1961. Immunodiffusion. New York and
London: Academic Press, 333 pp.

Crowle, Alfred J. 1963. Immunodiffusion - some consider-
ations in its use. J. Asth. Res., 1:11-38.

Evans, A. S., M. A. Stirewalt and M. MacKenzie. 1955.

Serologic reactions in Schistosoma mansoni infections.

 

II. Cercarial behavior in electrophoretically separated
fractions of sera of infected and uninfected mice. Exp.
Parasitol., 4:419-426.

Grabar, P., and C. A. Williams, Jr. 1953. Méthode per~
mettant l'étude conjugé des propriétés électrophorétiques
et immunochimiques d'un mélange de protéines; applica-
tion au sérum sanguin. Biochem. BiOphys. Acta,

10: 193-194.

87

Gray, George W. 1951. Electrophoresis. Sci. Amer., 185:
45-53.

Hardy, W. B. 1899. On the coagulation of proteid by elec-
tricity. J. Physiol., 24:288-304.

Heiner, D. C., and S. V. Kevy. 1956. Visceral larva mi-
grans. New Engl. J. Med., 254:630-636.

Heremans, J., J. Clausen, M. Th. Heremans, and R. Rask-
Nielsen. 1959. Immunoelectrophoretic characteristics
of mouse serums as basis for studying pathological
changes in serums of mice carrying transplantable
.malignant growths. J. Nat. Cancer Inst., 22:44-55.

Hirschfeld, Jan. 1960. Immunoelectrophoresis - procedure
and application to the study of group specific variations
in sera. Sci. Tools, 7:18-25.

Huntley, Carolyn C., and Alvin Moreland. 1963. Gel dif-
fusion studies with Toxocara and Ascaris extracts.
Amer. J. Trop. Med. Hyg., 12:204-208.

Jennings, R. K., E. Arditi, and M. A. Kaplan. 1960. Gel
diffusion studies of serologically related antigens. I.
Zone displacement in complex precipitating systems.
Int. Arch. Allergy, 17:257-266.

Jung, R. C., and G. Pacheco. 1958. Relationship of clinical
features to immunologic reactions in visceral larva mi-

grans. Amer. J. Trop. Med. Hyg., 7:256.

88

Jung, R. C., and G. Pacheco. 1960. Use ofa hemagglutina-
tion test in visceral larva migrans. Amer. J. Trop. Med.
Hyg., 9:185-191.

Kagan, Irving G. 1957. Serum agar double diffusion studies
with Ascaris antigens. J. Infect. Dis., 101:11-19.

Kagan, I. G., L. Norman, and D. S. Allain. 1959. Studies
of serology of visceral larva migrans. J. Immunol.,
83:297-301.

Kent, Naim H. 1960. Isolation of specific antigens from A_s_-

caris lumbricoides variety suum. Exp. Parasitol., 10:

 

313-323.

Korngold, L. 1956. Immunological cross-reactions studied
by the Ouchterlony gel diffusion technique. J. Immunol.,
77: 119- 122.

Korngold, L. 1961. Abnormal plasma components and their
significance in disease. Ann. N. Y. Acad. Sci., 94:
110-130.

Kraut, N. 1956. An electrophoretic study of sera from rats
artificially infected with and immunized against the

larval cestode Cysticercus fasciolaris. J. Parasit., 42:

 

109-121.
Kruidenier, F. J., and A. K. Katoh. 1959. Electrophoretic

studies of the sera of white rats and cats following

89

infection with Paragonimus kellicotti Ward, 1908, J.

 

Parasit., 45(Section 2):l7.

Kunkel, H. G. and A. Tiselius. 1951. Electrophoresis of pro-
teins on filter paper. J. Gen. Physiol., 35:89-118.

Kuttler, K. L., and D. W. Marble. 1960. Serum protein
changes in lambs with naturally acquired nematode in-
fections. Amer. J. Vet. Res., 21:445-448.

Lee, Hong Fang. 1960. Effects of superinfection on the be-

havior of Toxocara canis in mice. J. Parasit., 47:

 

583-588.

Leland, S. E., Jr., W. D. Lindquist, and H. A. Lillevik.
1955. An electrophoretic and chemical fractionation
study of sera from rats immunized against the nema-
tode Nippostrongylus muris. Exp, Parasitol., 4:208-225.

Leland, S. E., Jr., J. H. Drudge, and Z. N. Wyant. 1959.

Studies on Trichostrorgylus axei (Cobbold, 1879) . III.

 

Blood and plasma volume, total serum protein and elec-
trophoretic serum fractionation in infected and uninfected
calves. Exp. Parasitol. , 8:383-412.

Leland, S. E., Jr. 1961. Blood and plasma volume, total
serum protein, and electrophoretic studies in helminthic

diseases. Ann. N. Y. Acad. Sci., 94:161-182.

90

Lewis, Lena A. 1960. Electrophoresis in Physiology.
.. Second edition. Springfield, Illinois: Charles C.
Thomas. 120 pp,
Mauss, E. A. 1941. Occurrence of Forssman heterogenic

antigen in the nematode Trichinella spiralis. J. Im-

 

munol., 42:71-77.

Miller, L. L., and W. F. Bale. 1954. Synthesis of all plasma
protein fractions except gamma globulin by the liver. The
use of zone electrophoresis and Lysine -C 14 to define the
plasma proteins synthesized by the isolated perfused liver.
J. Exp. Med., 99:125-132.

Nichols, Robert L. 1956a. The etiology of visceral larva
migrans. 1. Diagnostic morphology of infective second
stage Toxocara larvae. J. Parasit., 42:349-362.

Nichols, Robert L. 1956b. The etiology of visceral larvae

migrans. II. Comparative morphology of Ascaris lum-

 

bricoides, Necator americanus, Strongyloides stercoralis,

and Ancylostoma caninum. J. Parasit., 42:363-399.

 

Oakley, C. L., and A. J. Fulthorp. 1953. Antigenic analysis
by diffusion. J. Path. Bact., 65:49-60.
Oliver-Gonzales, J. 1943. Antigenic analysis of the isolated

tissues and body fluids of the roundworm Ascaris lum-

 

bricoides variety suum. J. Infect. Dis., 72:202-212.

91

Oliver-Gonzales, J. 1944. The inhibition of human iso-

agglutinins by a polysaccharide from Ascaris suum.

 

J. Inf. Dis., 74:81-84.

Oliver-Gonzales, J. 1946. Immunological relationships among
polysaccharides from various infectious organisms. J.
Infect. Dis., 79:221-225.

Oliver-Gonzales, J. 1953. Absorption of A-2 isoagglutinogen
like substances of infectious agents on human isoagglutin-
ogens. J. Infect. Dis., 74:173-177.

Olson, LeRoy J., Barna Richard, and Adam Ewert. 1960. De-
tection of metabolic antigens from nematode larvae by
microculture agar-gel technique. Tex. Rep. Biol. Med.,
18:473-479.

Oshima, Tomoo. 1961. Standardization of techniques for in-

fecting mice with Toxocara canis and observations on the

 

normal migration routes of the larvae. J. Parasit., 48:
652-656.

Ouchterlony, O. 1953. Antigen-antibody reactions in gels.
Acta Path. Microbiol. Scand., 32:231-240.

Ouchterlony, O. 1962. Diffusion in gel methods for immuno-
logical analysis. Progr. Allergy, 6:30-154.

Oudin, J. 1952. Specific precipitation in gels and its applica-
tion to immunochemical analysis. Meth. Med. Res., 5:

335-378.

92

Perlmann, G. E., W. W. Glen, and D. Kaufman. 1943.
Changes in the electrophoretic pattern in lymph and
serum in experimental burns. J. Clin. Invest., 22:
627-633.

Peters, Theodore, Jr. and Christian B. Anfinsen. 1950.
Production of radioactive serum albumin in liver slices.
J. Bio. Chem., 182:171-179.

Pitts, T. D. 1962. Iii vitro culture of the larvae of Ascaris

 

lumbricoides variety suum. Continued studies with

 

newly hatched larvae. J. Parasit., 48 (supplement):37.

Porret, R. 1816. Curious galvanic experiments. Ann
Philosophy, 8:74-76.

Poynter, D., B. V. Jones, A. M. R. Nelson, R. Peacock,

J. Robinson, P. H. Silberman, and R. T. Terry. 1960,
Recent experiences with vaccination. Vet. Rec., 72:
1078-1090.

Proom, H. 1943. The preparation of precipitating sera for
the identification of animal species. J. Path. Bacteriol. ,
55:419-426.

Raffel, Sidney. 1961. Immunity. Second edition. New York:
Appleton Century-Crofts, Inc. 646 pp.

Sadun, E. H., L. Norman, and D. Allain. 1957. The detection

of antibodies to infections with the nematode Toxocara

93

c_a_rli_s, a causative agent of visceral larva migrans.
Amer. J. Trop. Med., 6:562-568.

Sadun, E. H., R. I. Anderson, and J. S. Williams. 1962.
Fluorescent antibody test for the serological diagnosis
of trichinosis. Exp. Parasitol., 12:423-433.

Scheidigger, J. J. 1955. Une micro -méthode de l'immuno-
électrophor‘ese. Int. Arch. Allergy, 7:103-110.

Schwonzen, T. 1951. IJber serumeinweissveranderungen bei
trichinose (unter beriicksichtingung der electrophorese) .
Klin. Wschr., 29:612-615.

Sharp, A. D., and L. J. Olson. 1961. Hypersensitivity

responses in Toxocara, Ascaris and Trichinella infected

 

 

guinea pigs to homologous and heterologous challenge . J.
Parasit., 48:362-367.

Shedlovsky, T., and J. A. Scudder. 1942. A comparison of
erythrocyte sedimentation rates and electrophoretic
patterns of normal and pathological human blood. J. Exp.
Med. , 75: 1 19- 126.

Silverman, P. H., D. Poynter, and K. R. Podger. 1962. Studies
of larval antigens derived by clutivation of some parasitic
nematodes in simple media: Protection tests in laboratory

animals. J. Parasit., 48:462-471.

94

Smith, M. H. D., and P. C. Beaver. 1953. Persistence and
distribution of Toxocara larvae in the tissues of children
and mice. Pediatrics, 12:491-496.

Smithers, S. R., and J. P. Walker. 1961. Serum protein changes
in monkeys infected with Schistosoma mansoni, with refer-
ence to metabolism of albumin. Exp. Parasitol., 11:39-49.

Soulsby, E. J. L. 1957. Antigenic analysis of Ascaris tissues
by the double diffusion precipitin test. Trans. Roy. Soc.
Trop. Med. Hyg., 51:9-10.

Soulsby, E. J. L. 1958. Studies on the heterophile antibodies
associated with helminth infections. I. Heterophile anti-

bodies in Ascaris lumbricoides infection in rabbits. J.

 

Comp. Path., 68:71-81.

Soulsby, E. J. L. 1960. Immunity to helminths - recent ad-
vances. Vet. Rec., 72:322-327.

Soulsby, E. J. L. 1961. Immune mechanisms in helminth in-
fections. Vet Rec., 73:1053-1058.

Soulsby, E. J. L. 1963. The nature and origin of the functional
antigens in helminth infections. Ann. N. Y. Acad. Sci.,
113:492-509.

Sprent, J. F. A., and H. H. Chen. 1949. Immunological studies

in mice infected with larvae of Ascaris lumbricoides. I.

95

Criteria of immunity and immunizing effect of isolated
worm tissues. J. Infect. Dis., 84:111-124.

Sprent, J. F. A. 1949. On the toxic and allergic manifesta-
tions produced by the tissues and fluids of Ascaris. I.
Effect of different tissues. J. Infect. Dis., 84:221-229.

Sprent, J. F. A. 1950. On toxic and allergic manifestations
caused by the tissues and fluids of Ascaris. II. Effect
of different chemical fractions on worm free, infected
and sensitized guinea pigs. J. Infect. Dis. , 86: 146-158.

Sprent, J. F. A. 1951. Toxic and allergic manifestations
caused by tissues and fluids of Ascaris. III. Hyper-
sensitivity through infection in the guinea pig. J. Infect.
Dis . , 88: 168- 177.

Sprent, J. F. A. 1952. On the migratory behavior of the
larvae of various Ascaris species in white mice. I.
Distribution of larvae in tissues. J. Infect. Dis. , 90:
165-176.

Sprent, J. F. A. 1963. Visceral larva migrans. Aust. J.
Sci., 25:344-354.

Stauber, Leslie A. 1954. Electrophoretic techniques. Exp.
Parasitol., 3:544-568.

Swartz, Benjamin. 1959. Evolution of knowledge concerning the

roundworm Ascaris lumbricoides. Smithsonian Report for

 

 

‘3"

96

1959, 465-481.
Taffs, L. F. 1960. Immunization of guinea pigs against Ascaris

suum. J. Helminth., 34:347-348.

 

Taffs, L. F., and A. Voller. 1962. Fluorescent antibody studies

i_n vitro on Ascaris suum. Goeze, 1782. J. Helminth.,

 

 

36:339-346.
Taffs, L. F., and A. Voller. 1963. 12 vitro fluorescent antibody

studies on Ascaris lumbricoides and Ascaris suum. Trans.

 

 

Roy. Soc. Trop. Med. Hyg., 57:353-358.
Tanner, C. E. and J. Gregory. 1961. Immunochemical study

of the antigens of Trichinella spiralis. Canad. J. Micro-

 

biol., 7:473-481.

Tiselius, A. 1937. Electrophoresis of serum globulin. II.
Electrophoretic analysis of normal and immune serum.
Bichem. J. , 31:313-317.

Turner, J. H. 1959. Experimental strongyloidiasis in sheep and
goats. 111. An attempt to induce passive immunity in lambs;
changes in serum proteins after infection; and effect of im-
mune serum on infective larvae. Proc. Helm. Soc. Wash.,
26(2):114-124.

Wadsworth, C. 1957. A slide microtechnique for the analysis of

immune precipitates in gel. Int. Arch. Allergy, 10:355-360.

97

Weber, T. B. 1957. Observations on the relation of serum
protein changes, antibody formation and eosinophilia in

cattle infected with the lungworm, Dicflrocaulus viviparus.

 

J. Parasit., 43 (Section 2):l4.

Wieme, R. J. 1959. An improved technique of agar gel elec-
trophoresis on microscope slides. Clin. Chim. Acta,
4:317-321.

Williams, Curtis A., Jr., and Pierre Grabar. 1955. Immuno-
electrophoretic studies on serum proteins. I. The anti-
gens of human serum. J. Immunol., 74:158-168.

Williams, Curtis A. 1960. Immunoelectrophoresis. Sci.
Amer., 202:130-140.

Wright, G. G., and J. Oliver-Gonzales. 1943. Electro-

phoretic studies on antibodies to Trichinella spiralis in

 

the rabbit. J. Infect. Dis., 72:242-245.