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Infection and Immunity, August 1999, p. 3947-3951, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Evidence of Thymus-Independent Local and Systemic
Antibody Responses to Cryptosporidium parvum Infection in
Nude Mice
Andrew A.
Adjei,
Janet T.
Jones,
Michael W.
Riggs, and
F. Javier
Enriquez*
Department of Veterinary Science and
Microbiology, University of Arizona, Tucson, Arizona 85721
Received 19 February 1999/Returned for modification 19 April
1999/Accepted 6 May 1999
 |
ABSTRACT |
Differences in susceptibility to persistent cryptosporidial
infection between two strains of adult athymic nude mice prompted us to
investigate the immune mechanism(s) that may control resistance to
infection in these T-cell-deficient mice. We studied fecal oocyst
shedding, serum and fecal parasite-specific antibody responses, and
fecal immunoglobulin levels in athymic C57BL/6J nude and athymic BALB/cJ nude mice following oral inoculation with Cryptosporidium parvum oocysts at 8 to 9 weeks of age. C57BL/6J nude mice had significantly higher fecal parasite-specific immunoglobulin A (IgA)
(days 27, 31, 35, and 42 postinoculation) and IgM (days 10, 17, 24, 28, 31, 38, 42, and 48 postinoculation) levels than BALB/cJ nude mice
(P < 0.05) and significantly higher serum
parasite-specific IgA levels at 63 days postinoculation
(P < 0.03). Moreover, C57BL/6J nude mice shed
significantly fewer C. parvum oocysts than BALB/cJ nude
mice from days 52 to 63 postinoculation (P < 0.05).
In contrast, BALB/cJ nude mice had higher levels of
non-parasite-specific IgA (days 38 to 63 postinoculation) and IgM (days
24, 35, 38, and 52 postinoculation) than C57BL/6J nude mice in feces
(P < 0.05). These data suggest that parasite-specific
fecal antibodies may be associated with resistance to C. parvum in C57BL/6J nude mice.
| |
INTRODUCTION |
During the past two decades,
Cryptosporidium parvum has gained recognition as an
important enteropathogen of mammals, including humans (17, 18,
48). In immunocompetent hosts, C. parvum generally
causes a self-limited diarrheal illness. However, in immunocompromised
hosts, C. parvum may cause a life-threatening, prolonged
cholera-like illness (12, 31, 37). Numerous attempts to
treat cryptosporidiosis in both humans and animals have met with
limited success (8, 41). The absence of consistently effective anticryptosporidial chemotherapeutic agents exacerbates the
consequences of this disease. Moreover, the lack of a suitable immunocompetent adult laboratory model of infection has hindered our
understanding of functional immune responses to this parasite.
The discovery that mice homozygous for the nude mutation
(nu/nu, T-cell deficient) and SCID mice (which lack both T
and B cells) develop persistent C. parvum infection led to
their use as models for the study of disease pathogenesis and
evaluation of potential anticryptosporidial agents (24, 32,
49). Evidence from the above studies and others (2, 10, 36,
50, 51) indicates that the control of cryptosporidial infection
in mice involves CD4+ cells and T-cell receptor (TCR)
CD4+ cells. Gamma interferon (IFN-
) is also
important in the control of parasite development, as treatment of mice
with anti-IFN- antibodies increases susceptibility to infection
(11, 47, 50). Serum and local parasite-specific antibodies
have been detected following infection, but their contribution to
resistance to cryptosporidiosis remains unclear (7, 44).
Several investigators have examined the kinetics of C. parvum-specific serum and fecal antibody responses associated with
oocyst shedding in infected mice, lambs, and calves. While some
investigators have found an association between declining oocyst
shedding and rising titers of specific immunoglobulin A (IgA) and IgM
(25, 38), others have provided no evidence for such an
association (44, 46).
Published studies on cryptosporidiosis in athymic nude mice have used
primarily the BALB/cJ strain; to our knowledge, little work has been
done with athymic nude mice of other inbred strains (24,
32). In our laboratory, inoculation of 8-week-old athymic C57BL/6J nude mice with C. parvum oocysts resulted in fecal
oocyst shedding significantly lower than that observed in athymic
BALB/cJ nude mice, suggesting non-thymus-dependent strain differences in susceptibility to infection. Recently, other investigators have
noted differences in susceptibility to cryptosporidial infection in
immunodeficient mice (13, 33, 47). These studies suggest that genetic differences, the presence or absence of certain cytokines (such as IFN- and interleukin-4 [IL-4]), and other host factors yet unidentified may influence the outcome of cryptosporidial infection. While cell-mediated immunity is required for control of
cryptosporidiosis, mucosally delivered neutralizing antibodies may be
involved in resistance to and recovery from infection (38, 40). We herein examine the relationships between local antibody (IgA and IgM) responses and oocyst shedding in adult C57BL/6J nude and
BALB/cJ nude mice. Our results indicate that parasite-specific serum
(IgA) and fecal (IgA and IgM) antibodies may be associated with
resistance to C. parvum infection in C57BL/6J nude mice. In
addition to providing insight into the role of anti-C.
parvum serum and fecal antibody responses, these two mouse
strains may serve as models to allow evaluation of thymus-independent
responses to C. parvum.
| |
MATERIALS AND METHODS |
Animals.
Female C57BL/6J nude and BALB/cJ nude mice (23 to
26 g), ages 8 to 9 weeks, were obtained from Jackson Laboratories
(Bar Harbor, Maine). Mice were housed in microisolator cages in
high-efficiency particulate air (HEPA)-filtered laminar flow racks and
allowed to acclimate for 1-week prior to oocyst challenge. All
manipulations were done in a HEPA-filtered hood. Mice received
sterilized normal mouse diet (Tekland, Harlan, Md.) containing 18%
(wt/wt) protein and 6% (wt/wt) fat as well as sterilized water
throughout the experimental period (63 days).
C. parvum oocyst inoculation.
Purified C. parvum oocysts (IOWA isolate) were obtained from Pleasant Hill
Farms (Mount Pleasant, Idaho). Oocysts were stored at 4°C in 2.5%
(wt/vol) potassium dichromate for up to 2 weeks prior to use. Before
mice were inoculated, oocyst preparations were treated with 1.75%
(wt/vol) sodium hypochlorite (1 min, 22 to 23°C) and then washed with
0.025 M phosphate-buffered saline (PBS; pH 7.2). Each mouse was
inoculated intragastrically with 106 oocysts in 100 µl of
PBS, using an 18-gauge gavage needle (Thomas Scientific, Swedesboro,
N.J.).
Quantification of oocysts in feces.
Fecal pellets from each
mouse in each group were collected twice weekly from days 3 to 63 postinoculation (p.i.) to monitor oocyst shedding by immunofluorescence
assay. Briefly, four fecal pellets were collected from each mouse into
individual microcentrifuge tubes and suspended in 1.5 ml of PBS
overnight at 4°C. The fecal pellets were then vortex homogenized and
centrifuged (1,500 × g, 4°C, 10 min), after which
the supernatant fractions were collected and kept at 
80°C prior to
specific IgA or IgM antibody measurements. Ethyl acetate (300 µl) was
then added to the fecal pellets in each tube; the feces were vortexed
(60 s) and then centrifuged (1,500 × g, 10 min, 4°C)
to concentrate oocysts. The supernatants were then aspirated, and the
oocyst-containing sediments were resuspended in 300 µl of PBS/sample.
Ten microliters of each suspension was then placed in individual wells
of multiwell glass slides previously treated with
poly-L-lysine (0.01% [vol/vol]; Sigma Chemical Co., St.
Louis, Mo.). Slides were heat fixed and processed for
immunofluorescence assay using oocyst-specific monoclonal antibody
(MAb) OW50 conjugated with fluorescein isothiocyanate as instructed by
the manufacturer (Meridian Diagnostics, Cincinnati, Ohio). Oocysts in
each sample were counted in an Olympus (La Palma, Calif.)
epifluorescence microscope (400× objective). Thirty arbitrarily selected fields in each well were examined.
Preparation of CpA.
C. parvum oocyst antigen (CpA) was
prepared by using a modification of a previously described protocol
(35). Briefly, oocysts (7 × 108) were
washed with PBS, centrifuged (1,500 × g, 5 min,
4°C), resuspended in 1.75% (wt/vol) sodium hypochlorite for 1 min,
centrifuged (1,500 × g, 5 min, 4°C), and washed
three times (4°C) with PBS. They were then resuspended in 2 ml of
sterile deionized water containing 1 mM phenylmethylsulfonyl fluoride
(Sigma) and disrupted by ultrasonication (10 min, 4°C) followed by
freezing and thawing five times. The lysate was then centrifuged
(1,500 × g, 10 min, 4°C) to remove insoluble
material; the supernatant was dialyzed (molecular weight exclusion
limit, 6,000 to 8,000) against PBS (12 h, 4°C) and then stored at
80°C. Protein content was determined by the bicinchoninic acid
assay (Pierce Chemical Co., Rockford, Ill.) according to the
manufacturer's instructions.
Fecal and serum antibody determination.
To determine the
local antibody response, four fecal pellets per mouse (collected from
days 3 to 63 p.i. from both strains of mice) were suspended in 1.5 ml of PBS, vortex homogenized, and centrifuged (1,500 × g, 4°C, 10 min) to remove insoluble material. The
antibody-containing supernatants were then collected and kept at
80°C prior to IgA and IgM determination by an indirect
enzyme-linked immunosorbent assay (ELISA). Each mouse was also bled
from the retro-orbital plexus on days 0, 3, 21, and 63 p.i. Serum
was separated and stored at 80°C prior to IgA, IgG, and IgM
determination by ELISA.
To measure C. parvum-specific antibody responses by ELISA,
Falcon Pro-Bind (Becton Dickinson, Mountain View, Calif.) ELISA plates
were coated overnight (12 h, 4°C) with 50 µl of CpA (2 µg/ml in
0.1 M carbonate buffer [pH 9.6]). The plates were then washed thrice
(5 min per wash) with PBS containing 0.05% Tween 20 (PBS-T) and twice
with PBS and then blocked (37°C, 1 h) with 100 µl of 1%
(wt/vol) nonfat milk in PBS; 50 µl of fecal supernatant (described
above) or serum (for IgA, 1:20 dilution; for IgG and IgM, 1:100
dilution in 1% nonfat milk-PBS solution) from individual mice was
then added in triplicate to wells and incubated (12 h, 4°C). Controls
included various concentrations of anti-C. parvum MAbs (IgA,
4D4 G9H4 [16]; IgM, OW50 [43]; IgG1,
3F7-F11 [42]). After washing, 50 µl of horseradish
peroxidase-labeled goat anti-mouse IgA, IgG, and IgM (1:1,000 dilution;
Sigma) was added to each well and incubated (37°C, 2 h). After
additional washing, ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic
acid)]-H2O2 (Kirkegaard & Perry Laboratories,
Gaithersburg, Md.) was added to each well and incubated (22 to 23°C,
30 min), after which optical densities (ODs) were measured at 450 nm
with an ELISA Reader (Dynatech Laboratories Inc., Chantilly, Va.).
Concentrations of parasite-specific serum IgA, IgG, and IgM in each
sample were calculated from positive controls determined on each plate.
Total fecal IgA or IgM levels were measured by sandwich ELISA using
various concentrations of class-specific antibodies as controls.
Capturing MAbs (Kirkegaard & Perry) included rat anti-mouse IgA
(catalog no. 01-18-01) and rat anti-mouse IgM (catalog no. 01-18-09).
Falcon Pro-Bind (Becton Dickinson) ELISA plates were coated with each
capture antibody (1 µg/ml in carbonate buffer [pH 9.6]; 12 h
at 4°C), washed thrice with PBS-T and twice with PBS as described
above, and blocked (1% [wt/vol] nonfat milk-PBS solution; 37°C,
1 h). Fecal supernatants and standards were then added in
triplicate to the wells. Positive controls used as standards consisted
of murine polyclonal antibodies derived from mouse myeloma cells (IgA,
MOPC-315; IgM, MOPC-104E; each at 0.0156 to 1.0 µg/ml; Sigma) and
were included independently in each ELISA plate. Plates were incubated
(12 h, 4°C), washed as described above, and incubated (37°C, 2 h) with 50 µl of horseradish peroxidase-labeled goat anti-mouse IgA
or IgM (1:1,000 dilution; Kirkegaard & Perry). After additional
washing, ABTS-H2O2 substrate (Kirkegaard & Perry) was added to each well and incubated (22 to 23°C, 30 min).
Following incubation, ODs were measured at 450 nm. The immunoglobulin
concentrations in each sample were calculated from immunoglobulin
standard curves determined on each plate.
Statistical analysis.
Parasite-specific fecal antibody and
total fecal immunoglobulin values for individual mice of each strain
were averaged, and differences between the means were analyzed for
significant differences by a random-effects repeated-measures model
using Stata Software (Computer Resource Center, Santa Monica, Calif.).
Differences in the mean serum anti-C. parvum IgA and IgM
antibody levels for each strain were analyzed for significant
differences by using the Wilcoxon rank sum test. Mean fecal oocyst
shedding results (days 3 to 63 p.i. from each strain) were
analyzed for significant differences by one-tail Student's
t test.
| |
RESULTS |
Oocyst shedding.
Oocyst shedding was undetectable in both
strains of mice from days 3 to 24 p.i. (Fig.
1). Variable, low-level oocyst shedding was noted in BALB/cJ nude mice from days 28 to 48 p.i. The
intensity of oocyst shedding in BALB/cJ nude mice increased at days 52, 56, and 59 and again at day 63 p.i. and was significantly higher than C57BL/6J nude mice oocyst shedding (P < 0.05).
Oocyst shedding in C57BL/6J nude mice was generally below detectable
levels before day 60 p.i., at which time transient oocyst shedding
was observed. The differential responses between C57BL/6J and BALB/cJ
nude mice described herein were observed in replicate experiments.
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FIG. 1.
Fecal oocyst shedding by C. parvum-inoculated
athymic C57BL/6J (n = 6) and BALB/cJ (n = 5) nude mice. *, values are significantly different
(P < 0.05).
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|
Fecal Cryptosporidium-specific antibody.
Although
there was considerable variability both between days and between
individuals within each group, on days 27, 31, 35, and 42 p.i.,
C57BL/6J nude mice had higher levels of anti-C. parvum IgA
than BALB/cJ nude mice (P < 0.05) (Fig.
2A). The titer of anti-C.
parvum fecal IgM was higher in C57BL/6J nude mice than in BALB/cJ
nude mice on days 10, 17, 24, 28, 31, 38, 42, and 48 p.i.
(P < 0.05) (Fig. 2B).
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FIG. 2.
Kinetics of fecal C. parvum-specific IgA (A)
and IgM (B) antibody responses in C. parvum-inoculated
athymic C57BL/6J (n = 6) and BALB/cJ (n = 5) nude mice measured by ELISA. *, values are significantly
different (P < 0.05).
|
|
Fecal immunoglobulin levels.
The kinetics of total fecal IgA
and IgM levels (Fig. 3) differed from
that of fecal C. parvum-specific antibody responses (Fig.
2). Total fecal IgA in BALB/cJ nude mice remained at a low level from
day 0 until day 31 p.i. On day 35 p.i., fecal IgA in BALB/cJ
nude mice increased sharply and remained significantly higher (days 38 to 63 p.i.) than in C57BL/6J nude mice (P < 0.05) (Fig. 3A). Conversely, total fecal IgA in C57BL/6J nude mice remained at a moderate level from days 0 to 35 p.i. (Fig. 3A). On days 17, 21, 24, 28, and 31 p.i., fecal IgA levels in C57BL/6J nude mice
were significantly higher than in BALB/cJ nude mice (P < 0.05) (Fig. 3A). Levels of fecal IgA in C57BL/6J nude mice dropped and remained significantly lower than those in BALB/c mice from days 42 to 63 p.i. (P < 0.001). The total fecal IgM in
C57BL/6J nude mice changed little over the course of the experiment
(Fig. 3B). However, in BALB/cJ nude mice, fecal IgM increased and was significantly higher than in C57BL/6J nude mice on days 24, 35, 38, and
52 p.i. (P < 0.05).
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FIG. 3.
Concentration of total fecal IgA (A) and IgM (B)
antibody responses in C. parvum-inoculated athymic C57BL/6J
(n = 6) and BALB/cJ (n = 5) nude mice
measured by ELISA. *, values are significantly different
(P < 0.05).
|
|
Serum Cryptosporidium-specific IgA, IgG, and IgM
levels.
On days 0, 3, and 21 p.i., anti-C. parvum
serum IgA was not detected in either C57BL/6J nude mice or BALB/cJ nude
mice (data not shown). On day 63 p.i., four of the five BALB/cJ
nude mice had no detectable anti-C. parvum serum IgA (Fig.
4). Anti-C. parvum serum IgA
in the one BALB/cJ nude mouse that responded was 44.1 µg/ml (Fig. 4).
In contrast, five of the six C57BL/6J nude mice showed a high level of
anti-C. parvum serum IgA ranging from 208 to 470 µg/ml
(Fig. 4). One C57BL/6J nude mouse had no detectable anti-C.
parvum serum IgA. The mean serum anti-C. parvum IgA
(BALB/cJ, 8.8 µg/ml; C57BL/6J, 310 µg/ml) on day 63 p.i. was
significantly different (P < 0.05) between the two
strains (Fig. 4).
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FIG. 4.
Concentration of serum anti-C. parvum IgA
antibody on day 63 p.i. in athymic C57BL/6J (n = 6) and BALB/cJ (n = 5) nude mice measured by
ELISA. Medians ( ; BALB/cJ, 8.82 µg/ml; C57BL/6J, 310 µg/ml) are
shown. Anti-C. parvum IgA antibodies in C57BL/6J nude mice
were significantly higher than in BALB/cJ nude mice (P < 0.03).
|
|
Anti-
C. parvum serum IgG levels at 3, 21, and 63 days p.i.
were highly variable between individual animals in both mouse strains.
There were no significant differences in serum anti-
C.
parvum IgG levels between BALB/cJ nude mice (day 3 p.i.,
16.71 ± 10.43;
day 21 p.i., 37.01 ± 21.58; day 63 p.i., 9.53 ± 5.19) and C57BL/6J
nude mice (day 3 p.i.,
8.841 ± 5.68; day 21 p.i., 30.22 ± 12.15;
day 63 p.i., 40.02 ± 33.54) at any time point studied (
P > 0.05).
Similarly, there were no significant differences in serum
anti-
C. parvum IgM levels between BALB/cJ nude mice (day
3 p.i., 5.00
± 1.58; day 21 p.i., 6.02 ± 0.72;
day 63 p.i., 2.24 ± 0.52) and
C57BL/6J nude mice (day 3 p.i., 3.841 ± 2.88; day 21 p.i., 9.65
± 4.82; day
63 p.i., 7.40 ± 3.37) at any time point studied
(
P > 0.05).
| |
DISCUSSION |
Athymic BALB/cJ nude mice have been used extensively as models of
C. parvum infection to investigate the role of humoral and cell-mediated immune responses (9, 24, 32, 41). However, to
our knowledge, no work on the immune response to C. parvum has been done with mice carrying the nu/nu mutation on other
strain backgrounds. Resistance to C. parvum requires both
cellular (primarily) and humoral responses (27, 41, 54).
These responses have been studied systemically in mice, humans, and
domestic animals. However, the role of intestinal mucosal immune
responses has not been thoroughly explored in athymic nude mice. During
our studies of mucosal immune responses, we observed that C57BL/6J nude
mice, despite the absence of functional thymus-dependent responses, develop only low-grade C. parvum infection, while BALB/cJ
nude mice exhibited greater infection. Specifically, BALB/cJ nude mice shed significantly more oocysts in feces than C57BL/6J nude mice. A
comparison of antibody responses between BALB/cJ nude mice and C57BL/6J
nude mice revealed marked differences. C. parvum-inoculated BALB/cJ nude mice had significantly lower production of fecal C. parvum-specific IgA (days 27, 31, 35, and 42 p.i.) and IgM (days 10, 17, 24, 28, 31, 38, 42, and 48 p.i.) than C57BL/6J nude mice, and only one of five BALB/cJ nude mice produced anti-C. parvum serum IgA. In contrast, BALB/cJ nude mice had significantly more total IgA (days 38 to 63 p.i.) and IgM (days 24, 35, 38, 48, and 52 p.i.) in feces than C57BL/6J nude mice, suggesting that in
BALB/cJ nude mice either secretory responses were not completely
impaired, there was increased leakage via increased intestinal
permeability, or parasite-specific responses were suppressed or not
appropriately expressed. Our results suggest that intestinal mucosal
immune responses temporally associated with fecal parasite-specific antibodies may accompany resistance to C. parvum infection
in C57BL/6J nude mice. Moreover, the results further suggest that differences in the two athymic nude mouse strains examined can have a
significant effect on susceptibility to C. parvum infection even though both strains are T-cell deficient. Recent reports have
demonstrated a relationship between oocyst shedding and local IgA
immune responses in C. parvum-infected lambs, calves, and mice (25, 38, 40, 46). In the experiments reported here, association of parasite-specific fecal IgA and oocyst shedding is also
observed in C57BL/6J nude mice.
Athymic nude mice lack functional systemic T lymphocytes
(34), have poorly developed or absent Peyer's patch
germinal centers, where progenitors of IgA plasma cells reside
(21, 29). These defects are corrected by adoptive
reconstitution with T cells (22). In addition, production of
IgA antibodies in mice requires the cooperation of cytokines, including
IL-4, IL-5, IL-10, IFN-, and transforming growth factor , which
have been found at low levels in athymic mice (14, 29, 30).
Recently, functional T lymphocytes at the mucosal level have been
detected in nude mice and identified as small intestinal intraepithelial lymphocytes (IEL), which are predominantly TCR 
IEL (23, 26, 52, 53). These T cells have been shown to promote mucosal IgA immune responses and have also been shown to
produce an array of Th1-type (e.g., IFN-), and Th2-type (e.g., IL-4,
IL-5, and IL-10) cytokines, as well as transforming growth factor and tumor necrosis factor alpha (3-6, 19, 30, 45). We have
found significantly higher numbers of TCR and IEL T cells
in the small intestines of C. parvum-inoculated C57BL/6J nude mice infected with C. parvum than in those of BALB/cJ
nude mice (1). The reasons for these differences between the
two strains of mice are difficult to explain. However, the putative lack of mature or functional IEL in athymic BALB/cJ mice may have contributed to the low fecal antibody responses observed. Although
the present study does not address whether IEL in C57BL/6J nude
mice are responsible for the lower oocyst shedding and increased serum
and fecal parasite-specific antibody levels observed, it is possible
that in the absence of systemic T cells, IEL had an effect on
either IgA+ B-cell differentiation, induction of IgA
switch, or other functional mucosal responses. Alternatively, it is
possible that IEL in C57BL/6J nude mice had influenced T
cells to up-regulate parasite-specific IgA and IgM mucosal responses
(15, 20, 39). Studies are now in progress in our laboratory
to explore these possibilities, and to define the factors influencing
development of mucosal and systemic antibodies in athymic C57BL/6J nude
mice and their potential involvement in the control of C. parvum infection.
| |
ACKNOWLEDGMENTS |
The excellent technical assistance of Kimberly M. Smith, Brian
Curran, and Vivian Mack is greatly appreciated. We also thank Kent
Griffith of the Arizona Prevention Center for help with statistical analyses.
This work was supported by Public Health Service grant AI 39203 from
the National Institutes of Health, Bethesda, Md.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Veterinary Science and Microbiology, Bldg. 90, Room 202, University of Arizona, Tucson, AZ 85721. Phone: (520) 621-4880. Fax: (520) 621-6366. E-mail address: fje{at}u.arizona.edu.
Editor:
T. R. Kozel
| |
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Infection and Immunity, August 1999, p. 3947-3951, Vol. 67, No. 8
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Abstract
Introduction
