B cell responses in the gut and mesenteric lymph nodes of mice to infection with Eimeria falciformis
by Patricia Varga Nash
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Veterinary Science
Montana State University
© Copyright by Patricia Varga Nash (1985)
Abstract:
The B cell response to infection with Eimeria falciformis (Eimer, 1870) Schneider, 1875, was
investigated in naive and immune mice after a single inoculation with the parasite. BALB/cByJ mice
were divided into three groups. Group 1 remained untreated throughout the study. Group 2 received
only one parasite inoculation of approximately.1000 oocysts on day 16, and Group 3 was given
approximately 1000 oocysts on day 0, and 1000 oocysts on day 16. Four mice from each group were
killed at 6 hr and 24 hr after inoculation on day 16, and then every other day through day 13
post-inoculation (PI). The large intestine, cecum and mesenteric lymph nodes (MLN) were collected
and processed for histological examination. An avidin-biotin immunoper-oxidase procedure was used
to stain for IgA-, IgM- and IgG-containing lymphocytes.
Primary and secondary IgA, IgM and IgG lymphocyte responses were seen in the large intestine of
non-immune (group 2) and immune (group 3) mice, respectively. IgA-containing lymphocytes were the
largest population of responding cells in this tissue. Cell numbers peaked 11 days PI in naive,
inoculated mice (group 2), and 9 days postchallenge (PC) in immunized, challenged mice (group 3).
IgM+ lymphocyte counts were highest 11 days PI in group 2 and 9 days PC in group 3, and IgG+ cell
numbers were greatest on days 11-13 PI in non-immune mice (group 2), and on. days 9-11 PC in
immune mice (group 3). There were no IgA or IgM responses in the cecum, but IgG+ cells exhibited
both primary and anamnestic responses to infection. There were primary but no secondary responses in
the MLN, and immunized mice had reduced IgA+ and IgG+ cell numbers in the nodes after challenge.
The largest population of responding cells in the lymph nodes contained IgG. Peak IgG and IgA
lymphocyte numbers were observed on day 11 PC in both groups of infected mice. IgM+ cell numbers
peaked on day 9 PC in both groups. IgA appears to be the most important antibody in the mucosal
response to E. falciform is infection, while IgG and IgM probably play a minor role in local immunity.
IgG may contribute substantially to the systemic response. B CELL RESPONSES IN THE GUT AND MESENTERIC LYMPH
NODES OF MICE TO INFECTION. WITH EIMERIA FALCIFORMTS
by
Patricia Varga Nash
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Veterinary Science
MONTANA STATE UNIVERSITY
Bozeman, Montana
December 1985
IV37?
N
1757
ii
APPROVAL
of a thesis submitted by
Patricia Varga Nash
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style, and consistency, and is ready for subm issio n to the
College of Graduate Studies.
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iii
STATEMENT OF PERMISSION TO USE
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requirements of master's degree at Montana State University,
I
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tha t
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Library
sha l l
make
it
available
to
borrowers under rules of the Library.
Brief quotations from
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without
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provided that accurate acknowledgment of source is made.
Permission for extensive quotation from or reproduction
of this thesis may be granted by my major professor, o;r in
his
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by
the
Director
of
Libraries
when,
in the
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opinion of either, the proposed use of the mat erial is for
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Any copying or use of the material in
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iv
ACKNOWLEDGMENTS
I
thank
my
major
advisor,
Dr.
C.A.
guidance and assistance with this work,
S p e e r , for
his
and the other m e m ­
bers of my graduate commit tee , Dr. Worley, Dr. Crowle and
Dr. Ewald, for their helpful discussions.
I also gratefully
ackno wl ed ge the contributions of Dr. Huffman of the M a t h ­
ematics D e p a r t m e n t for his assistance with the statistics
for
this
project,
and
Carol
Bittinger
of
Science Department for her help with the data
the
Computer
analysis
and
graphics.
Special
thanks go to Gayle Callis
of the Veterinary
Science Histology Laboratory for teaching me histotechnique,
and for her technical assistance.
I also want to a c k n o w l ­
edge the members of the Electron Microscopy Laboratory,
Kyle,
Bill
Whitmire
and David
teaching me techniques,
Jean
Reduker for their help
in
and I thank Merrie Mendenhall of the
Veterinary Science Photography Laboratory for the many fine
pictures illustrating this study,
and for her preparation of
/
slides for oral presentation of this work.
V
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS......................
iv
LIST. OF TABLES..................................
vi
LIST OF FIGURES..............
vii
ABBREVIATIONS........
ix
ABSTRACT.......... '.................... ....... ...........
.
%'
CHAPTER
1
INTRODUCTION................
I
2
LITERATURE REVIEW...................................
5'
3
MATERIALS AND METHODS.............................
15
Parasite......................... ................
Animals..... ..............................
Experimental protocol........... ... .
Tissue collection andprocessing................
Immunoperoxidase staining...................
Growth of IgG-secreting solid tumor...............
Statistical evaluation............................
4
I5
16
16
17
18
21
21
RESULTS.............................
Parasite stages.............................
Morphological changes in infectedmice............
Lymphocyte distribution
inthe g u t ............
Lymphocyte distribution in the mesenteric
lymph nodes..................................
IgA lymphocyte response...........................
IgM lymphocyte response.
42
IgG lymphocyte response........................
5
DISCUSSION. .......................................
6
SUMMARY............................
REFERENCES CITED
23
25
27
32 .
32
48
- 56
74
vi
LIST OF TABLES
Table
I•
2.
3.
4.
5.
6.
Page
IgA lymphocyte counts in the large intestine of
control mi ce , and in non-immune and immune
mice infected with E.. falciformis..............
36
IgA lymphocyte counts in the cecum of control
mice, and in non-immune and immune mice
infected with E.. f alciformis . .................
39
IgM lymphocyte counts in the large intestine of
control mice, and in non-immune and immune
mice infected with E.. falciformis..............
45
IgM lymphocyte counts in the cecum of control
mice, and in non-immune and immune mice
infected with E.. falciformis..............
48
IgG lymphocyte counts in the large intestine of
control mice, and in non-immune and immune
mice infected with E.. falciformis.............
50
IgG lymphocyte counts in the cecum of control
mice, and in non-immune and immune mice
infected with E.. falciformis...................
51
vii
LIST OF FIGURES
Figure
1.
Page
Tissues stained with the i m m u n o p e r o x i d a s e
procedure and used as assay controls.......
20
Developmental stages of, Eimeria fale iformis in
the large intestine and cecum ofmi ce ..........
24
Degenerate oocysts in the intestinal lamina
propria and lymphoid nodules of mice infected
with E i m e r i a f a l c i f o r m i s ...................
26 r
Erosion of the intestinal epithelium and
infiltration of cells into the lamina propria
in a non-immune mouse 9 days PI with E..
falciformis..................
27
Intestinal mucosa of a control mouse and an
immune mouse 11 days PC with £. falciformis.
Tissue stained for IgA+ lymphocytes..........
29
IgA-positive intraepithelial lymphocytes in the
epithelium over a lymphoid nodule in the large
intestine...................................; . . .
30
Large intestine of a control mouse and an immune
mouse 11 days PC with E_. f ale if ormis. Tissue
stained for IgG+ lymphocytes..................
31
8.
Mesenteric lymph
nodes
stained for IgA+
cells...
33
9.
Mesenteric lymph
nodes
stained for IgM+
cells...
34
10.
Mesenteric lymph
nodes
stained for IgG+
cells...
35'
11.
IgA lymphocyte response in the large intestine
of non-immune and immune mice infected with
Eimeria f ale if ormis . ............................
37
IgA lymphocyte response in the cecum of nonimmune and immune mice infected with E_.
f ale if ormis.....................................
38
IgA lymphocyte response in the mesenteric lymph
nodes of non-immune and immune: mice infected
with E .fale if ormis. Experiment I.............
40'
2.
3.
4.
5.
6.
7.
12.
13.
viii
LIST' OF FIGURES (continued)
Figure
14.
15.
16 .
17.
18.
19.
20.
21 .
22.
Page
IgA lymphocyte response in the mesenteric lymph
nodes of non-immune and immune mice infected
with E.. f ale if ormis . Experiment 2 ............
41
IgM lymphocyte response in the large intestine
of non-immune and immune mice infected with E .
falciformis.....................................
43
IgM lymphocyte response in the cecum of nonimmune and immune mice infected with E..
falciformis....................
44
IgM lymphocyte response in the mesenteric lymph
nodes of non-immune and immune mice infected
with E.. f a l c i f o r mis. Experi men t 1...........
46
IgM lymphocyte response in the mesenteric lymph
nodes of non-immune and immune mice infected
with E.. falciformis. Experiment 2 ............
47
IgG lymphocyte response in the large intestine
of non-immune and immune mice infected with E..
f alciformis.....................................
49
IgG lymphocyte response in the cecum of nonimmune and immune mice infected with E_.
falciformis.....................................
52
IgG lymphocyte response in the mesenteric lymph
nodes of non-immune and immune mice infected
with E_. falciformis. Experiment 2 ............
54
IgG lymphocyte response in the mesenteric lymph
nodes of non-immune and immune mice infected
with E.. f alciformis. Experiment I.............
55
ix
ABBREVIATIONS
ABC
avid in-biotin-peroxidase complex
clg
cytoplasmic immunoglobulin
DAB
diaminobenzidine tetrahydrochloride
HPF
high power field
IEL
intraepithelial lymphocytes
IFA
immunofluorescent antibody
-MeOH
methyl alcohol
MLN
mesenteric lymph nodes
PBS
phosphate buffered saline
PC
post-challenge
PI
post-inoculation
SRBC
sheep red blood cell
X
ABSTRACT
The B cell r e s p o n s e to i n f e c t i o n w i t h E i m e r i a
falciformis (Eimer, 1870) Schneider, 1875, was investigated
in naive and immune mice after a single inoculation with the
parasite.
B A L B / c B y J mice were divided into three groups.
Group I rem ained untreated throughout the study.
Group 2
received only one parasite inoculation of approximately .1000
oocysts on day 16 , and Group 3 was given approximately 1000
oocysts on day 0 , and 1000 oocysts on day 16 . Four mice
from ea.ch group were killed at 6 hr and 24 hr after ino cul a­
tion on day 16 , and then every other day through day 13
po st -i noc ula tio n (PI).
The large intestine, cecum and
me sente ric lymph nodes (MLN) were collected and processed
for histological examination.
An avidin-biotin immunoperoxidase procedure was used to stain for IgA-, IgM- and IgGcontaining lymphocytes.
Primary and secondary IgA, IgM and IgG lympho cyt e r e ­
sponses were seen in the large intestine of n o n - i m m u n e
(group 2) and im mu n e (group 3) mice, respectively.
IgAcontaining ly mph ocy tes were the largest population of r e ­
sponding cells in this tissue.
Cell numbers peaked 11 days
PI in naive, inoculated mice (group 2), and 9 days, p o s t ­
challenge (PC) in i m m u n i z e d , challenged mice (group 3).
IgM+ lymph ocy te counts were highest 11 days PI in group 2
and 9 days PC in group 3, and IgG+ cell numbers were g r e a t ­
est on days 11-13 PI in n o n - i m m u n e mice (group 2 ), and on
days 9-11 PC in immune mice (group 3). There were no IgA or
IgM responses in the cecum, but IgG+ cells exhibited both
primary and anamnestic responses to infection.
There were
primary but no secondary responses in the M L N , and immunized
mice had reduced IgA+ and IgG+ cell numbers in the nodes
after challenge. The largest population of responding cells
in the lymph nodes contained IgG.
Peak IgG and IgA lympho­
cyte numbers were observed on day 11 PC in both groups of
infected mice. IgM+ cell numbers peaked on day 9 PC in both
groups.
IgA appears to be the most important antibody in
the mucosal response to E. falciformis infection, while IgG
and IgM probably play a minor role in local immunity.
IgG
may contribute substantially to the systemic response.
I
CHAPTER I
INTRODUCTION
Coccidiosis
in
poultry
is
and
a clinically
livestock
severe
resulting
intestinal
from
infection
oocysts of one of several Sporozoan gen,era.
domestic
disease
with
Virtually every
animal will be exposed to these widespread coccid-
ian parasites during
its
lifetim e
(24).
Members
of the
genus E i m eria cause morbidi ty and high mor ta li ty in young
animals,
and are responsible for heavy losses in the cattle
and poultry industries.
It is estimated
that the annual
monetary loss in the United States due to this disease is at
least $90 million
ruminants
(24).
in dom estic birds,
Approximately
5-20%
and $30
of the
million
cattle
in
treated
for bovine cocccidiosis die from the infection (23 ).
Eimeria bovis
site that causes
weaned calves.
site
is
intestinal disease,
para­
particularly in
in
sufficient
n u m b e r s , as
in a feed lot
Severely infected animals exhibit characteristic
acute symptoms
orexia,
severe
intracellular
It also affects adult cattle when the para­
present
situation.
is a host-specific,
including copious
dehydration,
weight
and increased respiration.
loss,
hemorrhagic
fever,
diarrhea,
an­
weakness, ataxia,
The disease may be chronic, with
/
2
less debilitating
manifestations of the same symptoms, but
most deaths occur during the acute stages (88).
The pathological changes associated with bovine coccidiosis pri ma ri ly affect the large intestine and cecum (88 ).
The ep ith eli um erodes from the mucosal s u r f a c e , and large
hemor rh agi c
areas
necrotic tissue.
mucosa
occurs,
greatly
are
present
and
may
be
surrounded
by
Extensive leukocyte infiltration of the
and
the mesenteric
lymph nodes
are often
enlarged.
The sexual stages of E..
gametes,
bo v is (microgametes,
macro­
and oocysts) appear to do the most dam age to the
intestinal
mucosa
in cattle
(88 ).
It has been
observed,
however, that Eimeria falciformis causes mucosal destruction
in mice during the earlier asexual stages of merogony as
well (66 ).
Meronts of E.. bovis may also cause some erosion
of the epithelium.
Practical,
effective immunization against this parasite
is not yet available and the mechanism of acquired
ance is poorly understood.
resist­
Both antibody- and cell-mediated
im m u n i t y appear to be involved in parasite rejection, but
the role of each system is unclear.
Investigation of the B
cell response in mice to infection with Eimeria falciformis
(E i m e r , 1 870)
about
the
Schneider,
relative
classes in protection.
1 875,
importance
will
of
provide -information
the
various
antibody
Changes in im munoglobulin-containing
lymphocyte numbers in the gut and mesenteric lymph nodes may
3
indicate
sponse,
which
parasite stages
stimula te
the imm une r e ­
and knowledge of the B cell mechanisms
involved
in
resistance could lead to the d e v e l opm en t of an effective
vaccine for bovine coccidiosis.
The mouse-E.. falciformis host-parasite relationship
is
a useful model for bovine coccidiosis caused by E. 'b o v i s .
The course of infection, pathogenesis, and d e v e l opm en t of
immunity have been well documented in the mouse (16, 20 , 65 69, 8 2 , 1 1 5), and there are many parallels b e twe en the two
systems.
through
The
life
stages
cycles
of
of merogony
both
eimerian
and g ame to gon y
species
go
in epithelial
cells of the large intestine and cecum (16 , 30).
The subse­
quent release of oocysts from these cells results
in d e ­
squamati on
hosts.
There
and
extensive
mucosal
are differences between
meront stages,
damage
the species
in
both
in the number of
and the first asexual generation of E. bovis
occurs in endothelial cells of the central lacteals of the
small intestine (28) while this stage of E> fal cif or mi s is
found
in glandular epithelial cells of the large intestine.
However, the progression of the life cycle stages of sporogony,
merogony,
parasites
gametogony and oocyst formation in the two
is identical.
There are also si mila ritie s
induced in these hosts.
in the i m m u n e responses
Both cattle and mice develop p a r ­
tial immunity to reinfection after one exposure to E. bovis
or to E. falciformis. respectively (30, 67,
114).
There may
4
be some parasite de v e l o p m e n t
and oocyst shedding after a
second inoculation, but the clinical symptoms are inapparent
or very
mild,
and
oocyst
production
is sharply
reduced.
Resistance is incompl ete t h o u g h , and may be ove rco me by a
large parasite dose.
The objectives of this study were to characterize the
immunoglobulin classes of B cells in the normal BALB/c mouse
gut and mesenteric lymph nodes,
and to identify the subsets
of B lymphocytes responding in mice infected with E.. falci­
form iS.
The time course of changes in lymphocyte numbers in
both naive and previously infected mice were examined after
parasite
stage,
inoculation.
B cell number,
gut are discussed.
The
relationships
and morphological
among
parasite
alterations
in the
Investigation of immunity to an eimerian
parasite in a murine system may clarify the role of one of
the effector m e c h a n i s m s
responsible for resistance to E..
bovis.
It may then be possible to artificially augment the
natural
responses
to
protect cattle
effectively
disease.
I
from
this
5
CHAPTER 2
LITERATURE REVIEW
I m m u n i t y to eimerian parasites has been studied by a
number of research groups, but is still not well understood.
Much of the work has been done using chickens and the v a r ­
ious chicken coccidia.
very
important,
This particular host is economically
and so has been
the subject of numerou s
studies of naturally acquired resistance,
methods of artificially inducing immunity.
ological Iy similar to m a m m a l s
in that
antibody- and cell-mediated systems.
and of possible
Birds are immun­
they possess both
Chickens have a thymus
for T cell d e v e l op men t and a bursa for B cell maturation,
and, in addition are capable of making all of the classes of
mammalian antibodies except IgE (35).
Investigations of the
i m mu ne response to Eimeria in poultry,
and the studies in
m a m m a l i a n hosts, have been helpful in elucidating some of
the mechanisms involved in acquiring resistance to coccidia.
Species specificity of immunity
The Eimeria are species specific in both their ability
to infect an animal, and in the sti mulation of a parasitespecific im mu n e response by the host.
studies
of
host
specificity
(reviewed by McLoughlin,
62),
have
been
The most extensive
done
in
chickens
ruminants (reviewed by Levine
6
and Ivens,
5 3).
54) and rodents (reviewed by Levine and I v e n s ,
Eimeria tenella from chickens could not be success -
fully
transm itt ed
(86 ),
although
there
pheasants (26).
be
infectious
birds.
fect
to t u r k e y s , d u c k s , pheasants
have
been
conflicting
or quail
results
in
Vetterling (127) also found E.. tenella to
in chickens
but
not
in other
gallinaceous
Similarly, E.. acervulina from chickens did not i n ­
quail
Mammalian
(8 6 ,
125),
coccidia
also
pheasants
exhibit
(125)
or
turkeys
host specificity.
(117).
Eimeria
bovis and E.. cylindrica parasitize domestic cattle but will •'
not comp let e their life cycles in pigs or goats ( 129 ), and .
E.. f a l c i f o r mis of mice does not infect rats or dogs (77).
This high degree of selectivity between host and parasite is
likely the result of immunological as well as physiological
factors (102), since it can be circumvented through the use
of immunosuppressants (62 , 118).
The host i m mu ne response is also highly specific for
the immunizing species and strain of coccidia.
Vaccination
with one species of Eimeria does not usually confer protec­
tion
against other
eimerian
parasites.
Imm u n i z a t i o n
of
chickens with E.. tenella does not protect them from inf e c ­
tion with E. n e c a t r ix (45, 107 , 126), and vice versa.
sistance to other avian (17) coccidia,
Re­
and to the Eimeria of
rodents (7) and rabbits (5) has also been shown to be s p e ­
cies specific.
Strain specificity of the host response to
variants of the same eimerian species has been demonstrated
7
w i t h c h i c k e n c o c c i d i a (46, 55, 78), and may have i m p o r t a n t
consequences for vaccine development.
Immunity ts. specific parasite stages
Second generation meronts appear to be the most immuno­
genic stage of E. ten el I a in chickens (36, 37, 49), and can
provide protection from challenge when inoculated
rectally (39).
intra-
This has not been dem on str at ed with other
avian Eimeria. but it appears that sporozoites
and gameto-
cytes of chicken coccidia do not induce a strong protective
response in.the host (58 , 109).
Inoculation of first gener­
ation merozoi tes of E.. b o v i s into the cecum of cattle p r o ­
tects them from oral challenge with the same parasite (31),
providing
further evidence that it is the asexual stages
which are the most immunogenic.
The host response to an eim erian parasite occurs p r i ­
marily during the invasive sporozoite and merozoite stages.
After excystation,
sporozoites may be prevented from enter­
ing intestinal epithelial cells,
as suggested by studies on
JLimerJj-.a .nleschulz i in the rat (7 2 ) and E. tenella in the
chicken (4, 51).
cells
Sporozoites that do penetrate epithelial
in a resistant host may be prevented from developing.
There are reports of abnormal sporozoite morphology follow\
ing cell penetration in immu ne chickens (3 8 , 51, 126 ), and
this stage is inhibited
97).
Ha m m o n d
et
al.
in E i m e r i a -infected
(29)
proposed
that
rabbits
the
(76,
protective
8
response to E_. bovis
stages,
but
later
in cattle acted primarily on the sexual
found
that both
meronts
and
gamonts
were
a f f e c t e d (30, 31).
There are neutralizing antibodies to sporozoites and
merozoites in the serum of chickens infected with E. maxima
and E." tenella (34, 59,
104),
and the presence of both anti­
sporozoite and anti-merozoite
chickens
has
been
IgA in the ceca of immunized
demonstrated
(19).
Serum
IgG antibodies
to sporozoites, merozoites, and oocysts, and anti-sporozoite
and anti-merozoite IgA from the cecum were observed in mice
immunized with E. falciformis (128).
escent
antibody
(IFA)
technique
to
Using an immunofluorevaluate
serum
reactiv­
ity, Mesfin and Bel la my (70) reported that E. f ale i f o r mis
sporozoites and merozoites were more immunogenic than gametocytes and oocysts.
Thus,
there appear to be host responses
to every stage of the coccidian life cycle, but it is u n ­
clear whether any of the antibodies produced are protective.
Antibody production
Both agglutinins
and precipitins have been detected in
the sera of cattle inoculated with E.. bovis (2).
est
titers
of agglutinating
antibody
to merozoite
were observed 23-41 days post-inoculation,
The h i g h ­
antigens
and some lysis of
this parasite stage occurred at low serum dilutions.
were morphological changes
tions.
in the parasite
There
at higher dilu­
The amount of precipita tin g antibody to extracted
9
oocyst antigens also peaked 23-41
with E.. b o v i s .
days after inoculation
Studies with E i m eria of chickens (13, 34,
44, 52, 56, 6 1 , 92, 100) found serum agglutinins to spo r o ­
zoites
and
m e r o z o i t e s , and precipitins
oocyst antigens.
to merozoi te
and
Complement-fixing antibodies and precipi­
tins to oocyst antigens have been detected in E.. st i e d a i infected rabbit sera (32, 98, 99) and Rose (100) described
complement fixation with antibody to second generation mero­
zoi tes of E.. t e n e l l a .
There
is evidence that serum opsonizing antibodies are
also produced in infections with E i m e r ia.
observed
in E.. tenella-infected birds
(104),
They have been
and sporocysts
and sporozoites incubated with serum from immunized chickens
are more readily phagocytized by macrophages than untreated
parasites (41 , 87 , 10 3).
Until recently,
little work has been done on the gut
mucosal antibody response because of its relative inaccessi-‘
bility.
Early
att empts
to
show
the
parasites of extracts of the cecum,
unsuc cessful
(4,
34,
39).
some pa ras ite -sp eci fic
feces from
infected
Orlans
negative
effect
or its contents,
and Rose
on
were
(8 1) did find
IgG and IgA in saline extracts of
chickens,
and Movsesi jan
et al. (73)
also describe IgG in the cecal contents.
Further evidence of the importance of local production
of
IgA
was
E..
tenella
reported by
in
chickens.
Davis et al. (19),
They
found
large
working
numbers
with
of
10
I g A 7-Positive lym ph oc yt es
in the cecal tissue of infected
chickens, but very few IgM- or IgG-c on t a i n i ng cells.
IgA
was
and
also
the p re dom in ant
intestinal
secretions,
and
antibody detected
an anamnestic
in cecal
response
in these
IgA titers was observed after a second parasite inoculation.
Passive transfer of immunity
Complete immunity to oral challenge with Eimeria sp. is
not transferable with serum.
Some resistance to E. nie-
s_c.h.
U Izi and E.. maxima can be conferred to naive hosts by the
repeated
injection
chickens
(101,
of serum
105),
from
infected
respectively,
but
rats
most
( 111 ) or
attempts
to
passively immunize chickens, mice and cattle have failed (8 ,
22,
114,
125).
Other
investigations using bu rs ect om ize d
(111) or cyclophosphamide-treated (47) chickens suggest that
there
is
a role for
these deficient
reinfection.
ant ibo dy -me di ate d
animals
were
somewhat
protection,
more
since
susceptible
to
However, a cellular component of immunity also
appears to be of equal or greater importance (69 , 111, 112).
All
of
these
antibody
transfer
studies
involve
the
intravenous or intraperitoneal administration of a circulat­
ing antibody,
probably of the IgG class,
a parasite of the intestinal mucosa.
to protect against
This method of passive
immunization does protect well against an intravenous para­
site challenge (57), but is not as effective when the chall­
enge
is
given
orally.
Since
there
are
no
known
extraintestinal
stages
of most
sp., serum
transfer
might not be expected to provide much protection.
There
may be some serum leakage
Eimeria
into the mucosa due to the in ­
flammatory processes associated with cdccidial infections
( 108 ), but it is more likely that anti-parasite antibodies
of
the
IgA
class
are
stimul ate d
and
produced
locally.
Orlans and Rose (81 ) were able to confer some resistance to
chickens by injecting
immunized birds,
little
contact
IgA purified
from
the cecal
contents
but again the antibody probably had very
with
the
parasites
due
to
the
route, of
administration.
B c_gJlg jji normal gut and mesenteric lymph nodes
There are no studies which describe
immunoglobulin-containing
intestine.
However,
cells
in
murine
the
or
isotypes
bovine
of
large
the distribution of B cell immunoglob­
ulin classes in the normal mouse small intestine has been
examined (18, 122, 123).
Twenty-two percent of lamina pro­
pria
cytoplasmic
lymphocytes
contain
immunoglobulin
(clg),
and 96% of these B cells are of the IgA heavy chain isotype.
A p p r o x i m a t e l y 3.7% of the total population were IgM- and
IgG-containing
cells.
B lymphocytes with cytoplasmic antibody have been iso­
lated from the mesenteric lymph nodes (MLN) of BALB/c mice,
and characterized (25, 122 , 123).
these
cells
in normal
mouse
MLN
The total percentage of
ranges
from 0.18-2.1%,
12
57-80% of which contain IgA.
isotype,
Bet wee n 10-40% are of the IgM
and 2.1-10% are positive for IgG.
IgA lym ph oc yt e trafficking in the mouse gut and a s s o ­
ciated
lymph
studied using
r a d iol abe l Ied
lymphocyte and immunofluorescence techniques.
Antigen stim­
ulation
where
(121).
nodes
has been
is believed
precursors
to occur in the B e y e r ’s patches (15),
of
St imu lat ed
clgA
are
the
lympho cyt es
pre do min an t
travel
via
the
cell
type
lymphatic
system to the mesenteric lymph nodes where they mature and
proliferate.
They migrate from the efferent vessels of the
nodes into the thoracic duct circulation,
and are finally
carried back to the intestinal lamina propria by the blood­
stream.
plasma
Here they complete
cells
(50).
the differentiation
IgA-containing
cells
patches ac cum ula te in the spleen as well,
process
fr om
to
B e y e r ’s
but that organ
does not appear to be an obligatory part of the circulation
route (120).
Lymphocytes from the MLN (25, 63,
93,
110) and
intestinal mucosa (124) have been found to ho me to the gut
lamina propria in cell transfer studies in syngeneic mice.
Mattioli
populations
and
Tomasi
of intestinal
(60)
report
IgA cells
that
there
in neonatal
are two
mice,
large group with a half-life of approximately 4.7 days,
another smaller population with a longer,
half-life.
a
and
but undetermined,
The life span of IgA lym phocytes may be d i f f ­
erent in adult mice or in antigenically stimulated animals.
The
half-life
of
lymph
node
pla sm a
cells
in
rats given
13
antigen is approximately one-half the normal half-life (79).
B cell response to antigen
There is disagreement concerning the role of antigen in
mucosal lymphocyte responses.
Evidence for antigen-indepen-
dent (83) and antigen—dependent (42, 43, 80 , 93) cell migra­
tion to the gut has been reported, and it appears that both
mechanisms may be operative.
There is IgA lymphocyte accum­
ulation in the mucosa as a result of organ-specific homing,
and due to antigen-driven memory cell division (94 , 95).
The intestinal plas ma cell response of mice
to oral
admin is tr ati on of sheep red blood cell (SRBC) antigens is
p ri ma ri ly of the IgA isotype (3),
producing cells are also present.
although IgM-
and IgG-
Andre et al. (3) observed
peak numbers of IgA and IgM cells.on day 11 PI in n o n - i m m u n e
mice,
and
on
challenge.
primary
day
9 PC
in
The anamnes tic
response.
immune
mice
after
an
SRBC
response was greater than the
IgA-containing
plasma
cells
predominate
in the mesent eri c lymph nodes after oral presentation of
SRBC
antigens
(6 ),
and
IgM-containing
lymphocytes
were de ­
tected following a single SRBC inoculation.
Antigen form affects both the magnitude and the isotype
characteristics of the mucosal response.
Crude preparations
of cholera toxin and toxoid are more effective in eliciting
intestinal immunity in rats than are more purified forms of
the
antigen
(91).
Similarly,
BxuC-S-IjLa
SLsiLjLti-S-,
a
n
particul ate antigen,
is more i mmu no gen ic than ovalbumin,
which is a soluble substance (9).
Intraileal infusion of j3.
abortus results in the accumulation of IgA and IgM isotype
plasma cells in the ilea of sheep, whereas administration of
ovalbumin stimulates the production of IgG-containing lympho­
cytes (9).
Adams et a I . (I) report an increase in
IgG plasma cells in the
IgA and
intestinal lamina propria of sheep
infected with Xr..ic h Q gtrOJlgy..!us c o l u b r i f o r m i s , a nematod e
that may present both particulate surface antigens and solu­
ble proteins
products.
in the form
of enzymes
or excretory-secretory
15
CHAPTER 3
MATERIALS AND METHODS
Parasite
Eim$rl 8 £-3.1c ifOrinIg; originally
obtained
Ernst (Animal Parasitology Institute,
was
maintai ned
Fo ll o w i n g
by periodic
USDA,
passage
inoculation by gavage
from
John V.
Beltsville,
through BALB/c
with
MD),
mice.
approximately, 1000
oocysts, mice were placed in cages with wire mesh floors,
and feces
material
were collected
was processed
on days 7,
8 , and 9 PI.
Fecal
to remove the fresh oocysts by fil­
tering it through successively smaller wire mesh screens
(20, 48, and 115 mesh) into a collecting pan.
The filtrate
was then centrifuged for 10 min at 1500 rpm, and the s e d i ­
mented material mixed 1:2 with S h e a t h e r 's sugar solution,
and stirred for I hr.
The mixture was poured
into petri
dishes and allowed to stand, uncovered for 1/2 hr.
The top
of the dish was inverted and placed: on the solution for 1/2
hr,
then
removed
and
potassium dichromate.
the
oocysts
rinsed
off
with
2 .5 %
The last two steps were repeated.
The oocysts were sporulated in 2.5% potassium dichrom—
ate by aerating the solution for 3 — 5 days at room t e m p e r a ­
ture.
Sporulated oocysts were stored in the same potassiuum
dichromate solution at 4°C for no more than two months,
and
16
rinsed thoroughly with tap water before inoculation.
All
experimental mice received approximately 1000 oocysts/ 0.5 ml
tap water, by gavage,
for each scheduled inoculation.
Animals
Twelve-week
(Jackson
o l d , ag e-m atche d
Laboratories,
Bar
Harbor,
specific pathogen- free environment,
tana
State
Control
University
and infected
animal
female B A L B / c B y J mice
ME)
raised
in a
and housed in the Mon­
facility
mice were kept
were
during
the
study.
in separate rooms,
and
animals passing oocysts were placed in cages with wire mesh
bottoms to minimize the possibility of retroinfection.
were checked for coccidia by sucrose
Mice
fecal flotation prior
to use in an experiment.
Experimental protocol
Ninety-six mice were divided into three groups of 32
animals,
and given one of the following treatment schedules:
Group I
Control mice-
untreated throughout study
Group 2
Non-immune mice-
Group 3
Immune mice-
inoculation with parasite
on day 16
inoculation with parasite on
day 0 ,
inoculation with parasite
challenge on day 16
Four mice from each of these groups were killed at 6 hr
after inoculation on day 16 , and at I, 3, 5, 7, 9, 11, and
13 days PI.
The ex per ime nt was repeated six months later
with the non-immune and immune groups only.
17
Ijsgyg collection and processing
Mice were killed by cervical dislocation and the cecum,
large intestine and mesenteric lymph nodes removed.
The
intestine and cecum were pinned to a piece of cork, cut open
longitudinally,
and cleaned of fecal material with a cotton
swab
with saline.
moistened
The flattened
tissues and the
lymph nodes were fixed for 3 hr in Bouin's fluid and then
dehydrated overnight in 50% ethanol.
The large intestine
was wrapped into a "Swiss roll" (71), and all tissues were
placed in 70% ethanol for several hr.
processed
through
a series
Fixed tissues were
of alcohol
and
xylene baths,
infiltrated with Surgipath infiltration media,
in Surgipath embedding
and embedded
media (Surgipath Medical
Industries,
Inc., Northbrook , IL).
The
gelatin
tissues
to glass
were
sectioned
slides
coated
at 3 i<m,
with
and fixed
Histostik
(Accurate
Chemi cal and Scientific Corporation, W e s t b u r y , NY).
tions were air-dried,
decerated,
and stained
with
Sec­
with hematox­
ylin and eosin for examination of parasites and morphologi­
cal
changes,
or
the
i m m u n o p e r o x idase
identification
of B cells.
sections
mounted
Company,
were
Fairlawn,
NJ).
with
Following
Per mo unt
procedu re
dehydration,
(Fisher
for
the
stained
Scientific
18
Immunoperoxidase staining
An avid in -bio tin -pe roxid ase complex (ABC) method (40)
was used to stain tissue sections for the presence of IgA,
IgG,
and IgM-containing lymphocytes*
Rabbit anti-mouse IgA
primary antiserum was obtained from Miles Scientific (Naper­
ville,
IL) and was reconstituted,
aliquoted,
and frozen at -
20°C.
Heavy chain-specific rabbit anti-mouse
IgG and anti-
mouse IgM came from Jackson I m m u n o R e s e a r c h Laboratories,
I n c . (Avondale, PA).
IgM
antibodies,
Sodium azide was added to the IgG and
to a final
concentration of 0.1%.
antisera were kept refrigerated.
These
The anti-IgA was thawed
and diluted to a final concentration of 1:50, anti-IgG was
used at a dilution of 1:300, and anti-IgM at a concentration
of 1:800.
Blocking serum, biotinylated anti-rabbit IgG, and
the ABC reagent were provided by a Vectastain Kit (Vector
Laboratories, Burlingame,
CA).
The biotinylated
secondary
antibody was diluted to a final concentration of 1:200 for
the IgA stain,
am ino ben zi di ne
and
1:500 for the IgG and IgM stains.
te tra hy dro chlor ide
(DAB)
(Sigma
Di-
Chemical
Company, St. Louis, MO) and hydrogen peroxide were used to
develop the brown precipitate on positive cells.
The staining procedure was that outlined in the Vectastain Kit with the exception
chloride
in the ABC
of the use of 0.5 M sodium
reagent buffer
to prevent
non-specific
staining of mast cells (14, Vector Laboratories,
communication).
personal
Briefly, slides were washed in phosphalte
19
buffered saline (PBS), pH 7.6, for 5 min after dec'eration
and rehydration.
Diluted normal goat serum was
block Fc receptors,
applied
to
followed 20 min later by the application
of the diluted pri ma ry
antis eru m for 30 min.
The slides
were rinsed in PBS and sections were covered for 30 min with
the diluted biotinylated
in PBS,
secondary antibody.
After rinsing
the tissue sections were imm er sed for 30 min in a
bath of 0.3% hydrogen peroxide in MeOH, to remove endogenous
peroxidase activity.
Another PBS rinse was followed by the
application of the ABC reagent for I hr.
rinsed
for 5 min
in PBS,
The slides were
and the color was developed
on
positive cells by i m me rsi ng the slides for 3-4 min in the
DAB-hydroge n
peroxide
solution.
A tap
water
rinse
was
followed by counterstaining with hematoxylin.
Three immunoperoxidase control slides were stained each
day along with the experi men tal
tissues.
One section of
normal BALB/c mouse small intestine was treated with diluted
normal rabbit serum in place of the primary antibody.
controlled
for
any cross-reactivity
of normal
proteins.
Positive and negative control slides
This
rabbit serum
primary antiserum were also stained (Fig. I).
for each
Normal BALB/c
mouse lung was the positive control for anti-IgA,
and an
IgM-s ec ret in g mouse hy brido ma (generously provided by Dr.
Diane Brawner) that had formed a solid tumor,
negative control;
The same
IgM -se creti ng
positive control for the anti-IgM
was used as a
tumo r
antiserum,
was the
and an IgG-
20
Fig. I.
Tissues stained with the immunoperoxidase procedure
and used as as s a y c o n t r o l s . A. I g M - s e c r e t i n g
hybridoma stained with anti-IgM. B. IgG-secreting
m y e l o m a stained with anti-IgG.
C. BALB/c mouse
lung stained with anti-IgA.
x 250.
21
secreting
solid
tumor (MFC
11 OVAr plasma cell tumor line,
A m er ic an Type Culture Collection,
negative
control
(see
below).
Rockville,
The
MD) was the
IgG-secreting
tumor
served as a positive control for the anti-IgG antiserum, and
the IgM-secreting tumor was the negative control.
IgA antiser um
was
also tested
against
the
The anti-
IgG-secreting
tumor to rule out any cro ss-re activ ity with I g G , and less
than one percent of B cells
in the lung control
sections
stained positively with either anti-IgG or anti-IgM.
Growth of IgG-secreting solid tumor
The MFC 11 OVAr plasma cell tumor line was cultured in
sterile RPMI 1640 m e d i u m (GIBCO, Grand Island, NY) s u p p l e ­
mented
with
0 .2% (w/v) sodium bicarbonate,
st re pt omyc in (5 U / m l ), pH 7.4.
every other day.
and penicillin-
The cells were subcultured
Two female B A LB/c mice were inoculated
intraperitoneally with approximately 2000 tumor cells/ 0 .2 ml
sterile m a g n e s i u m -
and calc ium -f ree Hank's balanced salt
solution (GIBCO, Grand Island, NY).
in diameter,
weeks.
formed
The tumor
was
at the
site
removed
A solid tumor,
of injection
1-2 cm
after
two
and processed by the same
methods used for the other mouse tissues.
!
Statistical evaluation
Positive cells in the intestine and cecum were counted
in ten contiguous
microscope
fields
of longitudinally
sec­
tioned gut. . Only mucosal sections not immediately adjacent
L
22
to lymphoid nodules were selected.
(HPF) was 0.126 mm2,
made
in
the
lower
Each high power field
and counts of the large intestine were
2/3
of
that
tissue.
Ten
microscope
fields, 0.2 m m 2 , were counted for each section of lymph node
medulla.
The
count s/t iss ue
were
totaled
separately
for
each
a n i m a l , tr ans for med to the square root of the total count,
and analyzed by analysis of variance and Duncan's multiple
range
tests.
23
CHAPTER 4
■ RESULTS
Parasite stages
Parasites
were concentrated
in the cecum
and anterior
1/3 of the large intestine, particularly in the early stages
before oocyst formation.
Six stages of parasite development
were observed (Fig. 2).
Sporozoites were present at 6 hr
and 24 hr PI in n o n - i m m u n e
immune mice.
mice,
but only at 6 hr PC in
Due to the sampling schedule, first generation
merozoite s were not detected;
however,
second generation
me rozoi tes were observed in the n o n - i m m u n e group on day 3
PI.
None were seen in immu ne animals.
generation merozo ite s were present
fected
mice,
On day 5 PI, third
in both groups of in ­
and fourth generation merozoites
were seen on
day 7 P I only in n o n - i m m u n e mice.
Microgamonts and macrogamonts were present in the epi­
thelial cells of n o n - i m m u n e mice on days 7, 9, and 11 PI,
and in immune mice on days 3, 7, and 9 PC.
Oocysts appeared
on days 7, 9, and 11 PI in the n o n - i m m u n e group, but were
observed only on days 7 and 9 PC in the immune group.
were greater
immune mice.
numbers
of all parasite
stages
There
in the non-
24
Fig. 2.
Developmental stages of Eimeria falciformis in the
large intestine and cecum of non-immune BALB/cByJ
mice.
A. Sporozoite in an apical epithelial cell,
24 hr PI.
B. Second generation meronts at 3 days
PI.
C. Third generation meronts in epithelial
cells at 5 days PI.
D. Fourth generation meront
with radially arranged m e r o z o i tes, 7 days PI.
E.
Microgamonts with peripheral nuclei, and macrogamon ts w i t h da rk c e n t r a l areas, 7 da y s PI.
F.
Oocyst, day 7 PI. x 1050.
25
Degenerate oocysts were retained in the lamina propria
and in the lymphoid nodules even after the ter mination of
the
patent
period
(Fig.
and
3).
They
were
observed
in the perifollicular
in both
organized
follicles
nodules.
Non-immune mice had degenerate oocysts in the gut
on days 9, I I , and 13 PI.
areas of the
They were present in the imm une
group at all times sampled.
Morphological changes in infected mice
The mucosal ep ith eli um in the large intestine became
flattened and began to erode on day 5 PC in both infected
groups (Fig. 4). Although there was some regeneration in the
non-immune mice by day 9 PI,
the lamina propria was exposed
in portions of intestine on days 7, 9, 11, and 13 PI.
sion
never
extended
lamina propria,
deeper
than
the
apical
part
Ero­
of
the
and was always less severe in immune mice.
There was an increase in the size and cel lularity of
the intestinal lamina propria on days 9, 11, and 13 PI in
non-immune mice (Fig. 4).
This response was present in the
immune group on all days sampled.
Some of the infiltrating
cells were identified morphologically as neutrophils,
cytes,
and lymphocytes,
and B cells were demonstrated
mono­
with
the immunoperoxidase stain.
Mucosal regeneration was accompanied by an increase in
crypt length.
This change in height was observed on days 9,
26
Fig. 3.
Degener ate oocysts in the intestinal lamina p r o ­
pria, x 400 (A); and lymphoid nodules, x 160, inset
x 500 (B) of mice infected with E. falciform!^.
27
mm
I
Fig. 4.
Erosion of the intestinal epithelium and infiltra­
tion of cells into the lamina propria in a nonimmune mouse 9 days PI with £. falciform is. x 100.
11, and 13 PI in n o n - i m m u n e mice,
and on days 0, 7, 9, 11,
and I 3 PC in i m mu ne animals.
Lymphocyte distribution in the gut
IgA
I g A - c o n taining lympho cyt es occupied areas throughout
the
lamina
control
propria
mice.
in
the
cecum
and
large
intestine
of
IgA cell density (67.2-117.0 cells/10 H P F )
was higher than that of the other two imm un o g l o b u l i n (Ig)
classes.
They were evenly distributed from tip to base of
the mucosa in the large intestine,
but were more c o n c e n ­
trated toward the base in the cecum.
The increase in IgA
cells in the intestinal lamina propria of mice infected with
28
E. f alciformis
mucosa
(Fig.
was concentrated
5).
There was
primarily
at the tip of the
also some basal
infiltration
of
ly mph ocy tes , but this was not as marked.
No IgA cells were observed in an intraepithelial loca­
tion except in the epi th eli um over lymphoid nodules (Fig.
6).
Only occasional IgA-positive B cells were seen in the
epithelium,
and
their numbers did not increase
in infected
mice.
Large numbers of IgA cells were located in the lamina
propria adjacent to lymphoid nodules.
These were present in
similar numbers in all three groups of mice.
IgM
There were few IgM-containing B cells in the intestinal
or cecal lamina propria of control mice (0.0-2.8 cells/10
HPF).
These were distributed evenly from tip to base of the
mucosa in both control and infected animals.
intraepithelial
nodules,
IgM
cells
were
observed
Occasional
over
lymphoid
but as with IgA-containing intraepithelial lympho­
cytes (IEL), there was no increase above control in either
non-immune or immune mice.
Large concentrations
in mucosal
of IgM
lymphocytes
areas adjacent to lymphoid nodules
trol and infected mice.
were observed
in both con­
29
Fig. 5.
Intestinal mucosa of a control mouse (top) and an
i m mu ne mouse (bottom) I I days PC with £. £filciformis.
Large numbers of IgA lymphocytes are con­
centrated in the apical portion of the lamina pro­
pria in the infected gut. x 160.
30
Fig. 6.
IgA-positive intraepithelial lym phocy tes in the
epi th el iu m over a lymphoid nodule in the large
intestine.
x 400.
IgG
B
cells
containing
cyt oplas mic
IgG
were
located
throughout the lamina propria of both the cec um and large
intestine.
IgG cell density
in control
animals
(1.0-6.0
cells/10 HPF) was similar to that observed for IgM lympho­
cyte numbers.
However, unlike the IgM cells, there was a
greater concentration of IgG cells at the base of the mucosa
in the large intestine of infected mice (Fig. 7).
No d i s ­
tributional changes were seen in the cecum.
There were large numbers of IgG-positive lym phocytes
adjacent to lymphoid
nodules,
IgM-containing B cells.
as was observed
with IgA and
31
• • J!
*
•
.•
Fig. 7.
*
•i«
v
*
% T ..
V
/\
Large intestine of a control mouse (top), and an
im mu n e mouse ( b o t t o m ) 11 days PC with £. £fllcjLXPXEis.
IgG-positive cells accumulate at the base
of the mucosa in infected mice.
x 160.
32
Lymphocyte distribution in the mesenteric lymph nodes
Lymphocytes containing IgA, Ig M, and IgG were concen­
trated
control
in the medulla
of mesente ric
lymph nodes
and infected mice (Figs. 8-10).
in both
Occasional positive
cells were seen in the perifollicular areas in controls, and
non-immune and immune mice had higher numbers of Ig-containing lymphocy tes around the B cell follicles.
Few B cells
with cytoplasmic Ig were observed within follicles.
The pri ma ry
i m mu ne
cell
response
in the large intestine of non-
mice peaked on day I I PI at 2.5 times the control
number.
cells/10 HPF.
The
mean
control
cell
count
was
96.0
IgA
There was a steady increase in the number of
positive cells starting at day 7 Pl in the first experiment,
and a decline after day 11 PI (Fig.
11).
Cell numbers on
days 11 and 13 PI in the non-immune group were significantly
greater than control
in both experiments;
however,
counts
on days 7 and 9 P I were significantly higher only in the
first experiment (Fig.
There
testine
was
11, Table
a secondary
of mice
reexposed
I).
IgA response in the large in ­
to E.. f ale if or m i s . which
earlier and greater than the prim ary response.
numbers were observed on day 9 PC.
was
Peak cell
IgA-positive cell counts
steadily increased from 6 hr PC to day 9 PC, when they were
2.0-3.0 times the control level of 109.0 cells/10 HPF (Fig.
33
B
Fig. 8.
Mesent eri c lymph nodes stained for IgA cells.
A.
Control mouse.
B. Non-immune mouse 11 days PI with
E. X a l c i f p r mis. x 100.
34
Fig. 9.
Mesent eri c lymph nodes stained for IgM cells.
A.
Control mouse.
B. Non-immune mouse 11 days PI with
E. falciformis.
x Ioo.
35
Fig.
10.
Mesenteric lymph nodes stained for IgG cells.
Control mouse.
B . N o n - i m m u n e mouse 11 days
with E.
falcifo r m is.
x 40.
A.
PI
36
11).
A decline in cell numbers was observed after day 9 PC,
but co un t s on all days PC w e r e . s i g n i f i c a n t l y g r e a t e r than
the co nt r ol (Fig. 11, Tabl e I).
Table I.
IgA lym ph oc yt e counts in the large intestine of
control mice, and in n o n - i m m u n e and imm u n e mice
infected with E\_ falciformis in experiment 2.
CONTROL
NON-IMMUNE
IMMUNE
Mean cell
count + S.D.
Mean cell
count + S.D.
Mean cell
count + S *D •
0.25
67.2 + 16.8
89.0 .+ 36.8
129.0 + 16.5
I
117.0 + 39.5
97.8 + 3.5
129.5 + 36.3
3
74.8 + 35.3
78.5 + 12.2
159.2 + 77.6
5
88.5 + 30.8
113.0 + 16.1
195.5 + 84.3
7
71 .2 + 35.1
133.5 + 67.5
197.8 + 30.0
9
109.0 + 28.5
98.8 + 51.4
218.5 + 46.6
Time PC
(days)
11
96.0 + 7.1
239.8 + 91.6
175.8 + 50.2
13
70.0 + 11.2
187.8 + 57.6
154.8 + 28.6
There was no IgA cell response to the infection in the
cecum of n o n - i m m u n e and imm une mice.
Cell numbers in the
immu n e group of e xpe rim ent I were sig nif icantly different
from control
but there
None
of
counts on days 3, 5, 9, and
was
the
no peak
cecal
IgA
or discer nib le
cell
counts
13 PC (Fig.
trend
12),
in the data.
in expe rimen t
significantly different from the controls (Table 2).
2 were
37
4 0 0 -i
350-
300-
250-
200
-
150-
100-
9
10
11 12 13 14
Days Post—challenge
Fig.
11.
IgA lymphocyte response in the large intestine of
n o n - i m m u n e (O) and i m m u n e (O) mice in fe ct ed with
ji. falciformis in experiment I.
Control IgA cell
numbers are shown also (□).
38
220
200
-,
-
IgA C ells/10 HPF
180-
160-
140-
100-
7
8
9
10
11
12 13 14
Days Post—challenge
Fig.
12.
IgA lymphocyte response in the cecum of non-immune
(O) and i m m u n e (O) m i c e infected w i t h E.. f ale i formis in experiment I.
Control IgA cell numbers
are s h o w n also (□).
39
Table 2.
IgA ly mph ocy te counts in the cecum of control
mice, and
in non-immune and immune mice infected
with Eh_ falciformis in experiment 2.
CONTROL
Time PC
(days)
Mean cell
count + S. D.
110.2
0.25
NON- IMMUNE
Mean cell
count + SeDe
28.7
IMMUNE
Mean cell
count + SeDe
91.5 + 9.7
76.8 + 11.4
I
135.0 + 69.2
108.2 + 16.1
91 .5 + 20.4
3
102.0 + 16.1
76.0 + 17.2
95.8 ± 29.8
5
89.8 + 4.2
78.5 + 13.5
88.2 ± 20.3
7
108.8 + 18.0
98.0 + 17.4
115.5 + 34.5
9
97.5 + 11 .7
87.8 + 52.7
91 .2 + 45.8
11
91.5 + 31 .6
94.5 + 14.8
69.8 + 13.7
13
95.0 + 11.9
103.8 + 14.0
94.5 + 22.3
An IgA cell response was observed
in the mesenteric
lymph nodes of both n o n - i m m u n e and immune mice.
However,
there was no anamnestic response in the immune group as peak
counts were seen on the same day in both g r o u p s , and the
peak numbers of positive cells were lower in immune animals
than in non-immune mice (Figs. 13 and 14).
Non-immune mice
had peak IgA-positive cell counts that were 6.4-8.4 times
the control on day 11 PI.
11 was
Mean control cell numb er on. day
122.5 cells/10 H P F .
Cell numbers on day 9 P I also
were significantly higher for the non-immune group
in both
experiments, and in experiment 2, counts on days 7 and 13 PI
40
1400-1
1200 -
1000-
800-
600 -
400-
200
-
1 i1r
2
3
4
5
6
7
8
9
10
11 12 13 14
Days Post-challenge
Fig.
13.
IgA l y m p h o c y t e r e s p o n s e in the m e s e n t e r i c l y m p h
n o d e s of n o n - i m m u n e (O) and i m m u n e (<>) m i c e
inf ected w i t h E. f a Icif o r mis in e x p e r i m e n t I.
Control IgA cell numbers are shown also (Cl).
41
1400-1
1200 -
1000
-
800-
600-
400-
200
-
3
4
5
6
7
8
9
10
I 1 I 1
11 12 13 14
Days Post—challenge
Fig.
14.
IgA l y m p h o c y t e r e s p o n s e in the m e s e n t e r i c l y m p h
n o d e s of n o n - i m m u n e (O) and i m m u n e (O) m i c e
inf ected w i t h E.. f a l c i f o rmis in e x p e r i m e n t 2.
Control IgA cell numbers are shown also (□).
H2
were
greater
than
controls.
Immune
mice
had
peak
cell
counts of 3.5-5.6 times control ( 122.5-137.2 cells/10 H P F )
on
days
9-11
PC.
Counts
on
all
other days
PC
were
not
different from controls.
JLgM. JLvrophocvte response
There
was
no significant
increase
in the large intestines of infected
(Table 3).
However,
in IgM cell numbers
mice
in experim ent
both non-immune and immune
animals
I
in
exper im en t 2 showed a mucosal IgM cell response (Fig. 15).
Peak numbers in the non-immune group were observed on day 11
PI at 22.5 times the mean control count of 0.5 cells/10 HPF.
Mice that were i m m u n e to E,. f alc if orm is had peak counts of
14.8 times control (1.0 cells/10 HPF) on day 9 PC.
Counts
on days 7 and 13 PI in n o n - i m m u n e mice also were s i g n i f i ­
cantly greater than the c o n t r o l s , as were the cell numbers
in the im mu n e group on days 3, 11, and 13 PC.
None of the IgM counts in the cecum were different from
the
control
cell
numbers
in expe rimen t
I (Table
4).
In
experiment 2, counts in immune mice were different from the
controls only at 6 hr PC,
and non-immune mice had more IgM-
positive lymphocytes than controls on days 7 and 13 PI (Fig.
16).
Although
the peak number of positive cells was o b ­
served on day 11 PC in immune mice,
this was not s i g n i f i ­
cantly different from the control value.
43
i
I
I
I
r
1 I 1 I 1
Days Post-challenge
Fig.
15.
IgM lymphocyte response in the large intestine of
n o n - i m m u n e (O) and i m m u n e K>) mi c e in fe ct ed wi t h
£. f a l ci fo r m is in experiment 2.
Control IgM cell
numbers are shown also (□).
IgM Cells/10 HPF
44
I 1 I 1
Days Post-challenge
Fig.
16 .
IgM lymphocyte response in the cecum of non-immune
(O) and i m m u n e (O) mi c e infected w i t h E.. f a Ic i f o r m is in experiment 2.
Control IgM cell numbers
are s h o w n also (□).
45
Table 3.
IgM ly mph oc yte counts in the large intestine of
control mice, and in n o n - i m m u n e and imm un e mice
inf ected with EL. falciformis
in experiment I.
CONTROL
Time PC
(days)
NON- IMMUNE
IMMUNE
Mean cell
count + S •D #
Mean cell
count + S •D •
0.25
I .2 + 1.3
0.0 + 0.0
0.5 + 0.6
I
1.8 + 1.5
0.5 + 0.6
2.2 + 1.3
3
0.5 + 0.6
I .2 + 1.3
. 0.8 + 0.5
5
I .0 + 0.8
1.8 + 2.2
1.8 + 1.7
7
I .0 + 0.0
0.2 + 0.5
2.0 + 1.4
9
I .0 + 0.0
2.5 + 2.4
3.8 + 3.0
11
0.5 + 0.6
2.2 + 0.5
4.5 + 7*0
13
2.0 + 2.7
4.8 + 6.8
0.8 + I .0
Mean cell
count + SeDe
There were large differences between experiments in the
results of the IgM counts
in mesenteric lymph nodes.
In
ex pe ri men t I, all cell counts except those on day 11 PI in
the n o n - i m m u n e mice and day 7 PC in the imm u n e mice were
below
the control
numbe r of cells (Fig.
17).
Mesenteric
lymph nodes of both groups of infected mice in experiment 2,
h o w e v e r , showed
an increase
which peaked at 2.3 times
cells/10
in IgM-p ositi ve
lymphocytes
the control cell num ber
H P F ) on day 9 PC (Fig.
18).
(337-0
Counts on all other
days PC were not different from the controls.
46
500 -I
450-
400-
350-
300-
250-
200
-
1 1I1I1I1Ir
3
4
5
6
7
8
9
10
11 12 13 14
Days Post-challenge
Fig.
17.
IgM l y m p h o c y t e r e s p o n s e in the m e s e n t e r i c l y m p h
n o d e s of n o n - i m m u n e (O) and i m m u n e (O) m i c e
infecte d wi t h E. f a l c i f o r mis in e x p e r i m e n t I.
Control IgM cell numbers are shown also (□).
47
1200-1
1000-
800
-
600
-
3
4
5
6
7
8
9
10
11 12 13 14
Days Post-challenge
Fig.
18.
IgM l y m p h o c y t e r e s p o n s e in the m e s e n t e r i c l y m p h
n o d e s of n o n - i m m u n e (O) and i m m u n e (O) m i c e
inf ect ed wi t h E. f a l c i f o r m i s in e x p e r i m e n t 2.
Control IgM ceil numbers are shown also (□).
48
Table 4.
IgM l y m p h o c y t e c o u n t s in the c e c u m of c o n tr ol
mice, and in non-immune and immune mice infected
with Ejl falciformis in experiment I.
CONTROL
Time PC
(days)
NON-IMMUNE
IMMUNE
Mean cell
counts + S.D.
Mean cell
counts + S.D.
Mean cell
counts + S.D.
0.25
0.0 + 0.0
0.5 + 0.6
0.5 ± 0.6
I
2.8 + 2.4'
0.0 + 0.0
0.8 + I .0
3
0.0 + 0.0
0.2 + 0.5
0.2 + 0.5
5
I .5 + I .0
I .0 + 0.0
0.8 + 0.5
7
0.2 + 0.5
0.5 + 0.6
0.8 + 1.0
9
0.8 + 1.5
0.2 + 0.5
0.8 + I .0
11
0.5 + 0.6
0.0 + 0.0
0.5 + 0.6
13
0.2 + 0.5
0.8 + I .0
0.0 + 0.0
IgG lymphocyte response
The primary
and
secondary
IgG lymphocyte
responses
in
the large intestine peaked slightly later than the IgA and
IgM cell numbers in that tissue.
The highest cell counts in
non-immune animals were observed on days 11-13 PI,
5.4-7.1
times
(Fig. 19).
the
control
counts
(2.2-3.5
and were
cells/10 HPF)
Only day 13 PI was significantly above control
in experiment 2,
and none of the counts for non-immune mice
were different from control in expe rimen t I, although the
data
show
an apparent
days
11 and
increase
13 PI (Table 5).
in positive cell numbers on
Peak
IgG cell
numbers
were
49
IOOn
i 1I1i1r
Days Post-challenge
Fig.
19•
IgG lymphocyte response in the large intestine of
n o n - i m m u n e (O) and i m m u n e (<» mice in fe ct ed wi t h
£. falciformis in experiment 2.
Control IgG cell
numbers are shown also (□).
50
Table 5.
IgG l y m p h o c y t e c ou nt s in the large i n t e s t i n e of
c o n t r o l mice, and in n o n - i m m u n e and i m m u n e m i c e
infected with Ei. f alc’iformis in experiment I.
CONTROL
Time PC
(days)
NON- IMMUNE
IMMUNE
Mean cell
count + S •D •
Mean cell
count + S •D •
Mean cell
count + S •D •
0.25
2.0 + 1.4
2.5 + 1.3
4.0 + 0.8
I
11.5 + 15.7
2.0 + 0.0
5 ;2 + 1 .5
3
3.2 + 2.6
3.0 + 1.8
9,2 + 4.5
5
2.8 -j- 1.3
2.5 + 1.0
7.8 + 5.2
7
I .0 + 1.4
9,5 + 3.9
20.0 ± 6.5
9
1.8 + 0.5
7.8 + 6.2
29.2 + 2.1
11
3.5 + I .0
11.0 + 8.3
37.8 + 13.0
13
2.2 + 3.9
12.2 + 7.2
33.0 + 22.9
seen on days 9-11 PC in the large intestines of i m mun e mice.
The highest counts were 10.8-30.4 times the control values
of 1.8-3.5 cells/10 H P F .
IgG-positive l ymp ho cyt e numbers
were higher than control on days 3-13 PC in both experiments
(Fig. 19, Table 5).
There was also a primary and an anamnestic IgG response
in the ceca of mice infected with E.. falciformis.
While the
non-immune group in experiment I showed no significant diff­
erences in cell c o u n t s , there was a trend toward increased
numbers of positive cells on days 7-13 PI (Table 6).
mice
infected
with
the
parasite
in
experiment
Naive
2 had
51
Table 6.
IgG l y m p h o c y t e c o u n t s in the c e c u m of c o n t r o l
mice, and in non-immune and immune mice infected
with EX. falciformis in experiment I.
Time PC
(days)
CONTROL
NON- IMMUNE
Mean cell
count + SeDe
Mean cell
count ± S .D.
Mean cell
count + S .D .
IMMUNE
0.25
2.5 ± 1.7
0.8 ± I .5
2.2 ± 2.2
I
6.0 + 4.1
0.5 + 0.6
I .2 + 0.5
3
I .2 + 0.5
1.2 + I .0
4.0 ± 4.7
5
1.0 + 0.8
2.0 ± I .2
4.5 ± 1.3
7
1.2 ± 1.3
4.2 ± 3.2
11.5 ± 6.6
9
1.5 + 0.6
. 4.5 + 6.4
14.0 + 3.6
11
1.0 + 1 .2
3.8 + 2.4
4.8 + 4.3
13
I .2 + 0.5
3.2 + 2.2
2.0 + 0.8
—*
significantly higher IgG cell counts in their ceca on days
7, 11, and 13 PI, which peaked at 11.5 times control (1.0
cells/10 H P F ) on day 11 PI (Fig. 20).
IgG lympho cy tes
immune group,
6),
and
highest
steadily from day 3 PC
in the
and peaked on day 9 PC in experiment I (Table
on days
counts
increased
The num ber of cecal
9-1 I PC
were
in exp er imen t
9.0-9.3
times
2 (Fig.
control
20).
The
(1.0-1.5 cells/10
HPF).
The mesenteric lymph nodes of immune mice did not show
a classical secondary IgG lym ph ocyt e response after re e x ­
posure to the parasite.
The primary response peaked on day
52
'I'I1Ir
1 I 1 I 1
Days Post-challenge
Fig. 20.
IgG lymphocyte response in the cecum of non-immune
(O) and i m m u n e (O) m i c e infected w i t h E.. f al ci formis in experiment 2.
Control IgG cell numbers
are s h o w n also (□).
53
11 PI at 18.7-43.2 times control (87.0 cells/10 HPF)..
The
immune group also peaked on day 11 PC, at 38,0 times control
in exper im en t
however,
2 (Fig.
21).
Immune
mice
i n .exp er imen t
I,
had a peak count of 12,1 times control on day 9 PC.
This cell number was significantly lower than counts in the
n o n - i m m u n e mice on day 11 PI of the same exp er ime nt (Fig.
22 ).
54
5000-1
4000 -
3000 -
O) 2000-
1000-
1 I 'T ' I ' r
I'I'I
2
3
4
5
6
7
8
9
10
11 12 13 14
Days Post—challenge
Fig. 21.
IgG l y m p h o c y t e r e s p o n s e in the m e s e n t e r i c l y m p h
n o d e s of n o n - i m m u n e (O) and i m m u n e (O) m i c e
inf ected w i t h £. f a l c i f o r mis in e x p e r i m e n t 2.
Control IgG cell numbers are shown also (□).
55
2500-1
2000 -
1500-
Ol
1000-
500 -
T 1 I 1 I ' I ' I 1 Ht
3
4
5
6
7
8
9
10
11
12 13 14
Days Post—challenge
Fig. 22.
IgG l y m p h o c y t e r e s p o n s e in the m e s e n t e r i c l y m p h
n o d e s of n o n - i m m u n e (O) and i m m u n e (O) m i c e
inf ect ed w i t h E. f a l c i f o r m i s in e x p e r i m e n t I.
Control IgG cell numbers are shown also (□).
56
CHAPTER 5
DISCUSSION
The i m m u n e response to eimerian parasites appears to
involve both antibody- and cell-mediated mechanisms.
While
there is strong evidence to suggest that acquired resistance
is T cell dependent (69, 111, 112), substantial amounts of
parasite-spacific i m m u n o g l o b u l i n are also produced during
infection
§1,
92,
(2,
13,
98-100,
19,
104,
32,
34,
128).
44,
52, 56, 59, 61, 70, 73,
Unsuccessful
attempts
to
passively transfer complete protection with serum (8, 22,
101, 105, 111, 114, 125) or secretory (8 1) immunoglobulin,
and studies of B cell-deficient chickens (47,
111), have led
some investigators to conclude that antibodies are relative­
ly unimportant in the immune response (67, 106).
passive
transfer
ad m i n i s t r a t i o n
studies,
of
however,
antibodies
to
involved
a gut
All of the
the parenteral
parasite.
These ■
immunoglobulins would be expected to have limited access to
the intestinal mucosa,
In addition,
stantial
and therefore to the target antigens.
although other m e c h a n i s m s act to confer s u b ­
protection
in B cell-deficient birds,
normal
mals still exhibit greater immunity to challenge.
ani­
The pre­
sence of parasite-specific antibody at the site of infection
may be an important factor in reducing parasite numbers and
57
tissue
damage,
and
the
role
of
this
system
should
be
reexamined.
In this s t u d y , there were fewer E.. f alcif ormis p a r a ­
sites in the large intestine and cecum of immune,
challenged
mice than in the guts of non-immune,
infected mice.
zoites,
and
second generation
merozoites
merozoites
were not observed
fourth
Sporo­
generation
in immunized animals,
whereas
histological examination of non-immune mice showed the pre­
sence of sporozoites and second, third and fourth generation
merozoites.
The parasite dose
administered
was
relatively
low compared with inocula given in other studies (20, 6567), and there were few sporozoites and second generation
m erozoites visible even in n o n - i m m u n e mice.
stages
of JE. f ale if o r m iff were
present
testines and ceca of the immune group,
m erozoites were not detected.
sporozoites
and
mice indicates,
second
however,
no changes
the
large
in­
some sporozoites and
The absence of observable
generation
merozoites
in
immunized
that the immune response acts on a
very early stage of development.
found
in
Since later
Mesfin and Bell a m y (67)
in sporozoite penetration
and
development
in i m m u n i z e d mice challenged with E.. f ale if o r m i s , but r e ­
ported that first generation meronts were degenerate and the
numbers of all asexual and sexual stages greatly reduced.
The observation of gameto c y t e s
in immune
mice
3 days
PC
provides further evidence for an inhibitory mechanism acting
on the asexual stages.
M a c r o g a m o n t s and microgamonts. are
58
not normally present in non-immune mice until 6 days and 6.5
days
PI
respectively
(I 16),
so
the
gamonts
observed
in
i m m u n e mice at 3 days PC were likely arrested at an early
stage
of
stress
development
of
after
a challenge
inhibitory
factors
the
first
inoculation
acting
on
inoculation.
may
an
have
invasive
reduced
stage
of
The
the
the
parasite.
The length of time that g a m e t o c y t e s and oocysts were
present in imm u n e , challenged mice was shortened
pared with these stages
in non-immune
mice.
when com­
Sexual stages
were seen on days 7, 9 and I I PI in naive animals, whereas
they were observed only on days 7 and 9 PC in the immunized
group.
The protective response appears to reduce both the
number, of
another
parasites
and
study of i m m u n i t y
period of immune
the
duration
of
infection.
In
to E,. f a l c i f o r m i s . the patent
mice after a single challenge with 20,000
oocysts was 3-5 days shorter than that seen after a primary
s infection (67).
The
nodules
other
presence
of degenerate
of the large
investigators,
intestine
oocysts
has
although this
in the
not been
stage
lymphoid
reported
by
is frequently ob­
served in the lamina propria (65-67, 69) f o l l o w i n g inf e c ­
tion.
Degenerate oocysts were found in both follicular and
unorganized
lymphoid
tissue
in this
study,
and
probably
become trapped after being released from the overlying epi­
thelium.
Oocysts were surrounded by closely apposed cells,
59
but it is unclear whether these cells are giant cells,
appear
Speer,
to
surround
unpublished
oocysts
in
the
lamina
propria
which
(C.A.
observation).
Immune mice sustained less mucosal damage after chall­
enge than did the non-immune animals.
Although the course
of epithelial flattening and erosion was much the same in
both g r o u p s , fewer
immunized
mice
areas of the mucosa were
affected
in
and damage was confined prim a r i l y to the
epithelium and occasionally to apical portions of the lamina
propria.
An increase in crypt length and in the cellularity of
the lamina propria was observed in both naive and immunized
mice after infection.
In non-immune
mice,
regeneration of
the e p i t h e l i u m and the resulting increase in crypt length
was first detected at 9 days PI,
days 11 and 13 PI.
present
at
inoculation.
6 hr
and was also present on
In i m m u n e mice,
PC,
16 days
elongated crypts were
after
the
initial
This supports the findings of Mesfin,
and Stockdale (66),
parasite
Bellamy
who reported an increase in crypt length
on days 9-16 PI in n o n - i m m u n e
mice,
In this
s t u d y , in­
creased crypt length was also observed on day 7 PC in i m m u n ­
ized mice,
Thus,
two days earlier than in the n o n - i m m u n e group.
there was less damage and faster repair in those mice
with acquired resistance to the parasite.
There was also a
difference in the time of onset of cellular infiltration and
enlargement of the lamina propria between the two groups of
60
infected mice.
While the non-immune group showed no change
until 9 days PI, there was an increase in cell number and in
the size of the lamina propria at all times examined PO in
immu n e mice.
cytes,
Many of the infiltrating cells were l y m p h o ­
a significant proportion of which were IgA-containing
B cells.
effects
A reduction
in parasite numbers
and pathogenic
in i m p u r e mice may be related to the increase in
mucosal lymphocytes during the early steges of infection.
IgA-containing B cells were concentrated in the apical
portion of the lamina propria in infected
proportionally
mice,
the largest isotype population
(95%) and infected animals.
and were
in control
Since Eimeria falciformis para­
sitizes epithelial cells, the localization of IgA l y m p h o ­
cytes in the
tween
mucosal tip may provide greater contact b e ­
antibodies
and parasites.
epithelial cells for
secretion
IgA is transported
onto
the luminal
across
surface
of
the mucosa (10-12, 74, 96), and would thus have access to
parasites within these cells.
Once secreted,
this class of
antibody may also function in "immune exclusion" (33, 119)
by c o m p l e x ing with the surface of extracellular
stages
of the parasite.
Specific
IgA
invasive
antibodies
to the
surface of E_. falcif o r m i s sporozoites and m erozoites have
been demonstrated
(20,
128).
The p r e d o m i n a n c e of an IgA isptype response to i n f e c ­
tion with an intracellular gut parasite may prevent immuno­
logic ally- induced cytopathic effects.
Both IgG and IgM bind
61
compl e m e n t ,
but
IgA
does
not
fix
these
lytic
proteins.
There are changes in epithelial cell membrane proteins dur­
ing infection with JLLmgJZj-.9 necatr ix (85), and the presence
of parasite antigens on host cells has been d e m onstrated
after infection with other coccidia (75,
89,
113).
Comple­
ment-fixing antibodies to these altered proteins could lyse
epithelial cells,
Therefore,
causing further dam a g e to host tissues.
IgA antibodies may act to block the binding to
mucosal cells of other potentially destructive immunoglobu­
lin
classes.
The number of lymphocytes with cytoplasmic
normal
mouse gut was only 3.6% of the total of the three
i m m y n o g l o b u l ip classes examined.
small
IgG in the
increase
There was a relatively
in IgG-positive cells
throughput
the lamina
propria during infection, but most of the cells accumulated
at the base of the mucosa.
This localization away from the
epithelium may have two functions.
First,
it could reduce
the amount of complement-fixing antibody in the vicinity of
potentially vulnerable host cells.
As mentioned above, this
woyld prevent tissue damage resulting from the host immune
response to the infection.
Secondly,
IgG antibodies at the
base of the mucosa may help prevent further parasite in-vasion into the submucosa.
quire
epithelial
penetration
cells
j£. falciform is appears to r e ­
to c o m p l e t e
its development,
but
of other cells by the motile forms could cause
injury to underlying tissues.
62
IgM-p o s i t i v e
B
cells
made
up
the
smallest
portion
(1.4%) of immunoglobulin-containing lymphocytes in the large
intestine and cecum of control mice.
There was a very small
IgM cell response to the parasite in some infected animals,
but
no change
in the distribution
lamina propria.
This antibody
of these cells
in the
isotype appears to play
a
minor role ip the B lymphocyte response to E.. falciformis.
Occasional B cells with cytoplasmic immunoglobulin were
observed
in the IEL compartment over lymphoid nodules,
the numbers did not change in infected mice.
in other areas of the large intestine.
but
None were seen
Less than 7% of IEL
possess surface i m m u n o g l o b u l i n in normal mouse small i n ­
testine (84),
and most of the I EL express T cell antigens
(21), so it is not surprising that very few mature intraepi­
thelial
B cells were observed.
In addition,
it has been
reported that B lymphoblasts from the mesenteric lymph nodes
will home to the lamina propria, but will not migrate to an
intraepithelial
stock,
location
unpublished
(McDermott,
observation).
which mature and proliferate
after
stimul a t i o n
M.R. and
Plasma cell precursors
in the mesenteric
by the parasite
J. Bienen-
in the gut
lymph nodes
may not be
destined for the IEL compartment.
The majority of the clg+ lymphocytes in the mesenteric
lymph nodes were observed in the medulla, where they were
presumably moving out of the nodes via the efferent vessels.
During peak responses,
the sinuses were completely obscured
63
by
these
isotypes.
B c e l l s , particularly
those of. the IgG and IgA
Although the precursors of these cells prolifer­
ate in the organized follicular tissue,
the fixation method
used in this study removed most surface antigens, and thus
lymphoblasts possessing only surface immunoglobulin were not
detected.
There were both primary and secondary B c e l l •responses
to infection with E. falciformis in the large intestine.
the
three
immunoglobulin
isotypes
studied,
Of
IgA-positive
lymphocytes accumulated in the greatest numbers in both nonimmune and immune mice.
The cell counts were significantly
different from control by 7 days PI in non-immune animals,
and
remained
higher
through
day
13 PI.
The
anamnestic
response in immune mice peaked earlier and was greater than
the response observed in non-immune animals.
IgA cell nu m ­
bers on all days PC were greater than control,
and probably
accounted for much of the increase in cellularity seen in
the lamina propria.
The heightened secondary response is
likely due to the presence of memory cells, generated during
the
initial
infection,
parasite challenge.
which can respond
quickly
to the
They may reside in the mucosa after
initial formation, but are more likely to recirculate b e ­
cause of their small size (90).
ic challenge,
specific
When stimulated by antigen­
memory cells would extravasate,
de­
velop into plasma c e l l s , and b e c o m e lodged in the lamina
propria.
This may account for the rapid increase in IgA+
64
cell numbers in i m m u n e mice in this study as well as for the
a namnestic response in intestinal IgA titers seen.in mice
with acquired resistance to E.. falciformis (20).
The primary and secondary IgG and IgM lymph o c y t e r e ­
sponses in the large intestine were also significant, but
represented
ulated.
a small
fraction
of the B cells
that
accum­
The m a x i m u m number of IgG+ cells was only 15% of
the total of the IgA+ lymphocytes, and IgM+ cell numbers in
the
large
intestine
were only 5% of that total.
Both of
these isotypes of immunogIobuI in-containing cells exhibited
primary and anamnestic responses after antigen challenge,
so
it can be assumed that parasite-specific
m e m o r y cells of
these antibody classes are also generated.
Their importance
in local immunity, however, appears to be secondary to the
role
of
IgA.
IgA-preducing
l ymphocytes
are
the
largest
population of B cel] s elicited by the oral administration of
particulate antigens such as those of SRBCs (3), Brucella
abortus
(9) and Trichostronevlus colubriformls
(I),
whereas
IgG-containing cells seem to be induced by soluble antigens
(9).
Eimeria falciformis may present primarily particulate
surface antigens to which IgA and some IgG cells respond,
well
as
soluble
antigens
such
as
enzymes
or
as
excretory-
secretory products that might tend to elicit IgG lymphocyte
production.
The IgA and
IgM cell numbers
in the large intestine
peaked at the same time in n o n - i m m u n e mice, 11 days PI.
IgG
65
l y m p h o c y t e counts were greatest slightly later,
days PI.
at 11-13
There appear to be two pools of Peyer's patch IgA+
B cells that migrate to the mesenteric lymph nodes after
stimulation, and then home back to the small intestine.
One
matures and returns to the lamina propria within 48 hr, the
s e c o n d , larger population, matures in the spleen and takes
at least 5 days to complete the process (90,
120).
If these
m e c h a n i s m s operate in the large intestine,
and the latter
type of cell migration is primarily responsible for the IgA
lymph o c y t e a c cumulation during E. f a l c i f o r m is infection,
then it appears
that the asexual merozoite stages may be
eliciting the observed B cell response.
Increased
IgA cell
counts are first observed 7 days PI, 5 days follo w i n g the
appearance of the earliest merozoite stages.
The number of
IgA+ lymphocytes then begins to decrease after day 11 PI, 5
days following the development of gametocytes from the last
merozoite
stage.
This
is consistent with serum reactivity
studies that indicate that the asexual stages are the most
immunogenic
(70,
128).
The cecal lamina propria did not exhibit an IgA or an
IgM
B cell
secondary
response,
but
there
small
primary
IgG lymphocyte responses in this tissue.
f.alciformis
parasitizes
the cecum
intestine,
and
and
are histologically
mucosa
were
as
well
as
and
Eimeria
the
large
the structure of the cecal lymphoid nodules
similar.
Therefore,
the
in­
ability of the cecum to mount a response comparable to that
66
in the large intestine is somewhat unexpected.
lamina propria
is greatly
reduced
mice, and the organ is rudimentary.
However, the
in the ceca
of normal
Thus, there may be much
less organ-specific homing of lymphocytes to this tissue in
both naive and antigen-stimulated animals.
IgA and IgG lymphocyte numbers in the mesenteric lymph
nodes peaked 11 days PC in both groups of infected mice, and
IgM cell
counts
were greatest
9 days PC.
secondary B cell responses of any isotype,
mice actually had reduced
challenge.
responses
There
were
no
and the imm u n e
in this tissue after
M e m o r y cells should preferentially migrate to
the gut after generation in the mesenteric
lymph nodes (63,
64), so an anamnestic B cell response would not occur in the
nodes.
But,
the reason for the depression of B cell prolif­
eration in challenged,
i m m u n e mice is unclear.
the result of a decrease
gut.
mals
It may be
in antigenic s t i m u l a t i o n in the
If local immune responses in previously infected ani­
can
quickly
reduce
the parasite
load
after
challenge,
fewer cells in the lymphoid nodules will be stimulated,
and
fewer blasts will migrate to the MLN.
The
IgA
and
IgM
cell
responses
approximately the same magnitude.
in
the
MLN
were
of
The IgM cell .counts that
were below control in the first experiment may have resulted
from
a sampling
error,
since very little lymph node tissue
was available for examination in that study.
Almost 5 times
more IgG-containing l y mphocytes accumulated
in the nodes
67
than either IgA+ or IgM+ cells, but most of these did not
migrate
to the large intestine because
cells were observed there.
relatively
few
IgG+
Mature lymphocytes of this iso­
type probably remain in the MLN and secrete antibody into
the circulation,
partment.
rather than migrating to the mucosal c o m ­
Serum IgG titers
increase greatly
10-14 days PI
and continue to rise more slowly through day 28 PI in nonimmune,
testinal
infected mice (20).
stages
There are no reported extrain-
of Eimeria falciformis. so the function of
serum antibody to this parasite is unclear.
However,
the
p e r m e a b i l i t y of the gut mucosa increases during infection
with other ,Eimeria species,
and this may provide some con­
tact between serum antibodies and parasite antigens (108).
Antibody-mediated acquired resistance to Eimeria falci£p.r,flU §„ in the mouse primarily involves the production of two
immunoglobulin classes:
IgA and IgG.
IgA appears to be the
most important isotype in mucosal
immunity to the parasite
and
numbers
may
effects.
act
to
reduce
parasite
and
cytopathic
IgG+ lymphocytes are probably not as important in
local immunity, but are prominent in the systemic response.
The T cell system and other cellular effector mechanisms are
necessary for the development
of immunity
to coccidia
(69,
111, 112), but p a r a s i t e - s p e c i f i c antibodies, acting to o p ­
sonize or agglutinate the invasive stages,
tegral part of the response.
bers
of
IgA-containing
cells
may be an in ­
The appearance of large n u m ­
in
the
lamina
propria
of
68
infected mice suggests that this antibody class may function
in acquired resistance, and that more appropriate passive
transfer studies are needed to clarify the role of antibodymediated immunity to Eimeria infections.
I
69
CHAPTER 6
SUMMARY
Bovine
coccidiosis,
. .
caused
by
Eimeria
bovis r is
a
severe intestinal disease of cattle for which there is no
effective vaccine.
Study of acquired resistance in a mouse
model of this host-parasite relationship should clarify some
of the mechanisms of the antibody-mediated
fection.
response to in­
The B cell responses of immunized and unimmunized
mice to infection with Eimeria falciformis were investigated
following parasite inoculation.
Changes.in parasite number
and tissue morphology were also observed.
Twelve week old,
female BALB/cByJ mice were divided into three groups and
given
one of the foll o w i n g
treatments:
Group
I "control
mice" remained untreated throughout the study, Group 2 "noni m m u n e mice" were inoculated on day 16 with approx i m a t e l y
1000
E,. falcif o r mis
oocysts,
and
Group
3 "imm u n e
mice"
received ap p r o x i m a t e l y 1000 oocysts on day 0 and 1000 o o ­
cysts on day I6 of the study.
group were killed
days
cecum
I, 3,
and
processed
stained
5, 7,
6 hr after inoculation oh day 16, and on
9,
MLN were
for
with
Four mice from each treatment
11
and
13
removed,
histological
hematoxylin
PI.
fixed
The
large
in Bouin's fluid,
examination.
and
intestine,
eosin
for
Tissues
and
were
examin a t i o n
of
70
mo r p h o l o g i c a l changes and parasite stages,
biotin
i m m u n o p e r o x idase procedure for
lymphocytes
counted
with cytoplasmic
in 10 H P F /tissue,
IgA,
or an avidin-
identification
IgM and IgG.
of
Cells were
and totals were compared using
analysis of variance and Duncan's multiple range tests.
Few e r parasites of all stages were seen in immunized
mice,
and no sporozoites or second and fourth generation
merozoites
seen
were observed
in n o n-immune,
in this group.
infected
mice.
All stages were
There
was
also less
mucosal damage and more rapid epithelial regeneration in the
immune group,
and these animals had increased cellular in­
filtration of the lamina propria on all days PC, whereas the
non-immune mice showed this response only on days 9-13 PI.
IgA+ cells responding to infection with E. falciformls
accumulated
in the apical end of the lamina propria,
while
I g G - c o n t a i n ing l y mphocytes were more concentrated at the
base of the mucosa.
does
not bind
This distribution may allow IgA,
c o mplement,
greater
access
which
to parasitized
epithelial cells that could be damaged by IgG and c o m p l e ­
ment.
IgG may interact with soluble parasite products which
diffuse away from the epithelium,
that invade deeper tissue.
distribution with infection,
or may bind
to parasites
There were no changes
in IgM
and very few clg+ B cells were
observed in an intraepithelial location in either non-immune
or immune
mice.
Ig-containing cells of all three
isotypes
71
were concentrated
in the medulla of the m e senteric lymph
nodes.
There were primary and secondary IgA,
responses
in
the
large
intestine.
IgM and IgG cell
IgA+
cells
were
the
largest population of B cells in both control and infected
mice.
Cell
cells/HPF)
numbers
on day
peaked
I I PI
at
2.5
times
in n o n - i m m u n e
control
mice,
and
(96.0
2.0-3.0
times control (109.0 cells/10 H P F ) on day 9 PC in i m m u n e
mice.
In experiment 2, IgM cell counts in this tissue rose
to 22.5 times
control
(0.5 c e l l s / 10 H P F ) on day
Tl PI
in
group 2, and 14.8 times control (1.0 cells/10 HPF) on day 9
PC in group 3.
intestine
The numbers of IgG+ lymphocytes in the large
were greatest at 5.4-7.1 times control (2.2-3.5
cells/10 HPF) on days 11-13 P I in n o n - i m m u n e mice,
and at
10.8-30.4 times control (1.8-3.5 c e l l s / 10 HPF) on days 9-11
PC in immune mice.
No IgA or IgM lymphocyte responses were observed in the
cecum.
There were, however, both primary and anamnestic IgG
responses in this tissue.
trol
(1.0
control
These peaked at 11.5 times c o n ­
cells/10
HPF)
on
day
(1 . 0 - 1 . 5
cells/10
11
HPF)
PI
and
on
9.0-9.3
days
9-11
times
PC,
respectively.
There were prim a r y but no secondary responses in the
m esenteric
lymph nodes
of infected
mice.
IgA+ and
IgG+
cells had peak numbers on day 11 PC in non-immune and immune
mice,
while IgM+ lymphocyte numbers
increased to 2.3 times
72
control 9 days PC in both groups.
IgA and IgG cell counts
were actually ]ower in immune mice on the peak day,
perhaps
due to the smaller number of parasites presenting antigen in
the large intestines and ceca of these animals.
IgA+ cell
counts were 6.4-8.4 times control (122.5 cells/10 H P F ) on
day
I I PI
in n o n - i m m u n e
mice,
and
3.5-5.6
(122.5-137.2 cells/10 HPF) in immune mice.
times
control
Peak IgG+ lymph­
ocyte numbers were 18.7-43.2 times control (87.0 cells/.10
H P F ) in non-immune, infected mice and 12.1-38.0 times c o n ­
trol in immunized, challenged mice.
IgG-containing cells
were the largest lymph o c y t e population in the MLN of both
groups of infected animals.
IgA appears to be the most important antibody in local
immunity to Eimeria falciformis. while IgG may play a larger
role in the systemic response to infection.
circulating antibody on intestinal parasites
therefore,
greater
The effect of
is minimal,
stimulation of mucosal immunity probably provides
protection.
73
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