MICROBIAL ECOLOGY IN HEALTH A N D DISEASE
VOL. 2: 47-59 (1989)
Characterisation of Protective Antibodies in Hamsters
Immunised Against Clostridium diflcile Toxins A and €3
PYEUNG-HYEUN KIM and RIAL D. ROLFE*
Department of Microbiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
Received 9 August 1988; revised 17 October 1988
Toxigenic Clostridium dtficile is the major cause of antimicrobial agent-associated pseudomembranous colitis in
humans and of ileocaecitisin golden Syrian hamsters. The pathogenicity of C. dt@ciZe is believed to be dependent on the
production of two immunologically and biochemically distinct toxins: toxin A (enterotoxin) and toxin B (cytotoxin).
In earlier investigations we demonstrated that hamsters immunised against toxins A and B are protected against
clindamycin-induced C. dtficile-associated ileocaecitis and that this protection is transferred to infant hamsters
through maternal milk. In the present investigation, the class-specific immunoglobulin response in adult and infant
hamsters immunised against C. djicile toxins A and B was examined using enzyme linked immunosorbent assays.
Parenteral immunisation of adult hamsters against toxins A and B induced IgG, IgA and IgM antibodies in hamster
sera specific for these two toxins. IgG antibodies to toxins A and B predominated in the milk and intestines of adult
hamsters immunised against toxins A and B and in the sera and intestine of infants from immunised adult hamsters. No
significant IgA or IgM antibodies to toxins A and B were detected in maternal milk and intestines or in infant sera and
intestines. IgG antibodies to toxins A and B were detected in infant caecal contents up until 10 d and in sera up until
approximately 24 d of age. The results from this investigation suggest that serum derived IgG antibody to C. djicile
toxins is primarily responsible for protection of adult and infant hamsters against C. dzficile-associated ileocaecitis
when immunised parenterally against toxins A and B.
WORDS-clostridium
Cytotoxin.
KEY
dzficife; Toxin A; Toxin €3: Antibody; Immunisation; Hamster; Ileocaecitis; Enterotoxin;
INTRODUCTION
Clostridium dificile-induced intestinal disease in
humans is a health problem of significant clinical
importance. Toxigenic C. dificile is the sole cause of
antibiotic-induced pseudomembranous colitis, a
severe and life-threatening intestinal disease, and is
the cause of approximately 20 per cent of cases of
antimicrobial agent-associated non-specific colitis
and diarrhoea without c o l i t i ~ .All
' ~ major classes of
antimicrobial agents have been reported to induce
C. dijicile-associated intestinal disease in humans.
In addition, C. dijicile is the cause of certain
intestinal diseases not related to antimicrobial therapy.13.14,24.37 The mechanisms by which C. dificile
induces intestinal disease are poorly understood.
However, C. dificile-associated intestinal disease
appears to involve many parameters including both
the virulence of the microorganism (e.g., toxin production) and its interaction with the physiological
status of the host (e.g., antibody).
*Authorto whom correspondence should be addressed.
089ILo6OX/89/01oO47-13$06.50
0 1989 by John Wiley & Sons, Ltd.
The pathogenicity of C. dificile, at least in part, is
related to the production of two biochemically and
immunochemically distinct toxins, 1 , 2 1 , 3 1 referred
to as toxin A, an enterotoxin, and toxin B, a
cytotoxin. It is not clearly understood which of the
two toxins is responsible for the pathological
changes seen in C. dificile-induced intestinal disease, though some evidence implicates both toxins.
For example, both toxins are consistently detected
in faecal specimens from humans and experimental
animals with C. dificile-associated intestinal disIn addition, the intestinal pathology and
death caused by purified toxins A and B in hamsters
mimic the pathology observed during the natural
disease
On the other hand, there is
evidence to suggest that toxin A plays a more
important role in the disease process than toxin B.
Mitchell et aLZ3 have shown that the secretory
and tissue damaging properties of C. dificile broth
filtrates on rabbit ileum are due to toxin A and not
toxin B. It has also been reported that purified
48
P.-H. KIM AND R. D. ROLFE
toxin A, but not toxin B, causes severe epithelial trate from a non-toxigenic strain of C. dijicile were
cell necrosis with destruction of villi and poly- not protected. Antibody against the immunising
morphonuclear infiltration in rabbit ileal loops.36
toxoid could be demonstrated in the serum, milk
Investigators have postulated that the ability of and caecal contents of hamsters. Protection against
humans to produce toxin-neutralising antibodies C. dzficile-induced intestinal disease could be
may not only influence the severity of C. dificile- passively transferred from immunised mothers to
associated disease, but also give protection against their infants, and neutralising antibodies to toxins A
further infection with this microorganism. Circu- and B could be demonstrated in infant serum and
lating antibodies to C. dzficile toxins can be intestinal contents. Foster mothering experiments
demonstrated in humans and an antibody response demonstrated that maternal protection of infants
frequently occurs in patients with C. dzficile- against C. dificile-associated ileocaecitis was transassociated d i a r r h ~ e a . ~For
. ~ ~example, 82 per ferred to infant hamsters primarily through breast
cent of individuals over the age of two years have milk and not across the placenta.
antibody to toxin A and/or toxin B, showing seroIn the present investigation, the class-specific
conversion occurs early in infancy.38 Analyses of immunoglobulin response in adult and infant
serologic responses among patients with C. dzficile- hamsters immunised against C. dificile toxins A and
induced intestinal disease show that approximately B was examined.
50 per cent of patients with C. dzficile-associated
diarrhoea develop a serum antibody response to one MATERIALS AND METHODS
or both of C. dzficile toxins during the d i s e a ~ e . ~ . ~
Furthermore, high antibody titres to C. dificile Maintenance of Hamsters
toxins correlate to a less severe clinical course of
Golden Syrian hamsters (Sasco, Inc., Omaha,
i n f e ~ t i o n . ~Other
,~
than the indirect evidence NE) were used throughout the course of this investidescribed above, there is no definitive data to show gation. Adult ( 23 mths of age) and infant hamsters
that circulating antibody is protective against C. were housed in conventional rooms and maintained
dzficile-associated intestinal disease in adults.
ad libitum on Purina Laboratory Rodent Chow
The Syrian Golden hamster has been used exten- 5001 (Ralston Purina Co., Richmond, IN) and
sively as an animal model in the study of C. dzficile- water. Hamsters were randomly assigned to treatassociated disease for several reasons.6 First, treat- ment or control groups. Female hamsters were bred
ment with antimicrobial agents induces an intestinal by housing one female and one male hamster
disease in hamsters similar to pseudomembranous together for 7 d after which the females were caged
colitis in humans. Second, human-derived isolates individually. All infants in a litter received the same
of C. dificile cause disease in hamsters. Third, C. treatment.
dzficile toxins appear to play a major role in the
pathogenesis of the disease in both humans and
Purzjication and Characterisation of Clostridium
hamsters. For example, in 1979, A110 et at.’ demonstrated that passive immunisation with Clostridium difficile Toxins A and B
sordellii antitoxin, which cross-reacts with C. dzfiClostridium dificile toxins A and B were purified
cile toxins, protected hamsters from clindamycin- using previously established techniques. 17,21,3
induced ileocaecitis and death. Much of what is The purification steps included preparation of a
presently known about the pathogenesis, epidemi- cell-free dialysate, ultrafiltration to collect material
ology, treatment and prevention of C. dzficile- > 100,000 molecular weight (Mr), Sepharose CL-6B
associated intestinal disease was originally disco- chromatography to collect Mr size fractions of
approximately 500,000, two different steps of
vered in hamsters and later confirmed in humans.
In earlier investigations, we examined the role of fractionation on DEAE Sepharose CL-6B and
antibodies against C . dificile toxins A and B in finally an acetate precipitation step on the toxin
protecting adult and infant hamsters against C. A preparation. Protein concentrations were deterdzficile-associated ileocaecitis.” Adult female mined using the protein-dye binding assay of
hamsters immunised parenterally with toxoid A or Bradford6 available from Bio-Rad Laboratories
a mixture containing both toxoids A and B were (Richmond, CA). Bovine gamma globulin was used
protected against clindamycin-induced C. dificile- as the standard.
associated fatal ileocaecitis. On the other hand,
The homogeneity of purified preparations of C.
hamsters immunised with toxoid B or a broth fil- dificile toxins A and B were characterised by
ANTIBODIES TO C. DIFFICILE TOXINS A AND B
49
polyacrylamide gel electrophoresis (PAGE) in 7.5 Collection of Hamster Specimens
per cent native gels” and Western blot analy~es,~’
Two weeks after completion of immunisation,
crossed-immunoelectrophoresis,40 cytotoxin neu- female hamsters were mated with non-immunised
tralisation, and enzyme linked immunosorbent
male hamsters. The infant hamsters were removed
assays with affinity purified rabbit antibodies to
from their mothers 6 d post-partum, sacrified, and
toxins A and B. At the final stages of the purification
intestinal homogenates and serum prepared from
process, the purified toxin preparations contain
each infant. Maternal milk was collected from each
only one (toxin A) or a few (toxin B) major compofemale hamster using a negative pressure suction
nents. This indicated that the toxin preparations
apparatus 24 h later.’ The female hamsters were
were substantially enriched and free of major levels
bled by cardiac puncture and serum prepared, and
of contaminants. More importantly, the purified
then sacrified and their intestinal tracts removed.
toxin A preparation did not contain detectable
Intestinal homogenates were prepared by homogetoxin B and vice versa.
nising the intestinal tracts (tissue and contents) in
equal volumes (w/v) of PBS. The homogenates were
centrifuged and the supernatants filter sterilised.
Immunisation of Hamsters Against Toxins A and B
Isolation of Immunoglobulin A (IgA) from Hamster
Female adult hamsters were immunised with Milk
toxoid A, toxoid B, a mixture containing toxoids A
Hamster IgA was isolated from the collected milk
and B (toxoid AB) or a broth filtrate prepared from by a modified method of Bienenstock.’ Twenty ml
a non-toxigenic strain of C . dzjicile (NTBF). of milk was mixed with 10ml normal saline and
Toxoids were prepared as described by Libby et centrifuged at 100,000 x g for I h at 4°C. After
al.” Briefly, toxoids A or B were prepared by the centrifugation, the clear aqueous layer between the
addition of 1 ml(O.5 mg) of purified toxins A or B to upper fatty layer and the pellet was collected. The
9ml (6mg of protein) of NTBF. Toxoid AB was solution was brought to p H 4 with I M HCI and
prepared by the addition of 0.5 ml of purified toxin centrifuged at 30,OOOxg for 30min at 4°C. The
A (0.5 mg) and 0.5 ml of purified toxin B (0.5 mg) to pellet (mostly casein) was discarded and the super9ml (6mg of protein) of NTBF. The NTBF was natant neutralised with 2 M Tris and centrifuged
prepared from a 72 h dialysis bag broth culture of again at 30,000 x g for 30 min at 4°C. The superC. dijicile VPI 2037 as described previo~sly.’~ natant was passed through a 0.45pm filter.
To each preparation, 0.1 ml of 40 per cent (v/v) Twenty-six ml(35 mg of protein) of the filtrate were
formaldehyde was added and the preparations were applied to a Sephadex G-200 column (2.5 x 120 cm)
incubated for 36 h at 37°C. Formaldehyde-treated which had previously been equilibrated with 0.1 M
NTBF was made by the addition of 1 ml of 50 mM sodium phosphate buffer (pH 6.8). The filtrate was
phosphate buffered saline, pH 7.2 (PBS) to 9 ml eluted with the same buffer at 4°C. The effluent was
(6 mg of protein) of NTBF and then treated with monitored for optical absorbance at 280 nm, and
formaldehyde as described above for toxoids A and 12ml fractions collected. The first major peak,
B.
which eluted at the void volume, was pooled and
Hamsters, weighing 70 to 80 g, were immunised concentrated by ultrafiltration. The retentate
subcutaneously once per week for 4 wks with 0.2 ml contained primarily IgA and was characterised by
of a 1:l mixture of Freund’s complete adjuvant ELISA.
(GIBCO Laboratories, Chagrin Falls, Ohio) and
toxoid A, toxoid B, toxoid AB or the NTBF.23 The
hamsters were next immunised subcutaneously once Isolation of Immunoglobulin M (IgM) from
per week for six weeks with 0.2 ml of a 1:1 mixture of Hamster Serum
Freund’s incomplete adjuvant and the toxoid prepAdult hamsters were bled by cardiac puncture.
arations. Animals were bled by cardiac puncture Hamster IgM was isolated from blood by a modi. ~
30 ml of blood
7 d after the final immunisation. Serum anti-toxin fied method of C O ~Approximately
titres were determined by using a cytotoxicity neu- was allowed to clot for 1 h at room temperature and
tralisation assay performed in HeLa tissue culture then overnight at 4°C. Fifteen ml of serum was colcells and enzyme-linked immunosorbent assays lected and dialysed against 2 mM sodium phosphate
buffer (pH 6.0) for 3 d at 4°C. The dialysate was
(ELISAs).
50
centrifuged at 2,OOOxg for 10min at 4°C. The
supernatant was discarded and the precipitate was
re-suspended in cold phosphate buffer and re-centrifuged. The washing was repeated once. The final
euglobulin precipitate was dissolved in 8ml of
0.1 M PBS (pH 8.0) and dialysed against 1 litre of
the same buffer overnight at 4°C.
Eight ml (35mg of protein) of dialysate was
applied to a Sephadex G-200 column (2.5 x 120 cm)
previously equilibrated with 0.1 M PBS (pH 8.0).
The sample was eluted with the same buffer at 4°C.
The effluent (7.5 ml/fraction) was monitored for
optical absorbanceat 280 nm. Those fractions which
eluted at the void volume were pooled and concentrated by ultrafiltration. Six ml(9 mg of protein) of
the concentrated retentate were re-applied to a
Sephadex G-200 column and eluted with 0.1 M
PBS (pH 8.0). Two ml fractions were collected and
each fraction which eluted at the void volume was
characterised by ELISA.
Characterisation of Mouse and Hamster ClassSpecijic Immunoglobulins
The specific reactivities of goat anti-mouse
IgG, IgA and IgM against mouse and hamster
class-specific immunoglobulins were examined
by enzyme linked immunosorbent assays. One
hundred microlitres of serial two-fold dilutions of
mouse or hamster class specific immunoglobulins
were added to each well of a 96-well microtitre plate
(Immunolon 11; Dynatech Laboratories, Inc.,
Alexandria, Va.). The mouse immunoglobulins
were purchased from commercial sources; IgG and
IgM were obtained from Calbiochem (La Jolla, CA)
and IgA was obtained from Sigma Chemical Co. (St
Louis, MO). Hamster IgG was purchased from PelFreez Biologicals (Rogers, AK). Hamster IgM and
IgA were prepared as described above. After incubation overnight at 4"C, the wells were washed five
times with 200 pl of PBS containing 0.05 per cent
Tween (PBST). After washing, 0.1 ml of goat antimouse IgG (y chain specific), IgA ( a chain specific),
or IgM (p chain specific) was added to the wells. All
antisera were purchased from Sigma Chemical Co.
The plates were incubated for 1 h at 37°C. The wells
were then washed and rabbit anti-goat IgG alkaline
phosphatase conjugate (Sigma Chemical Co.) was
added to each well and incubated for 1 h at 37°C.
After washing five times, 0.1 ml of phosphatase
substrate (1 mg of p-nitrophenyl phosphate, disodium, per ml dissolved in diethanolamine buffer,
pH9.8) was added to each well, and plates were
P.-H. KIM AND R.D. ROLFE
incubated for 30 min at room temperature. The
reaction was terminated by the addition of 20 pl of
5 N NaOH to each well. The optical density at A,,,
was measured in a microplate colorimeter.
ELISA for Detection of Hamster Antibodies to
Toxins A and B
Purified toxins A and B were used to prepare
ELISA plates for detecting antibodies to toxins A
and B in hamster sera, intestinal homogenates, and
maternal milk. C. dijicile toxins A and B were each
diluted to a final concentration of 0.5 pg/ml in PBS.
One hundred microlitres of the diluted toxin were
added to each well of a 96-well microtitre plate.
After incubation overnight at 4"C, the wells were
washed five times with 200 p1 of PBST. Serial twoor five-fold dilutions of the materials to be tested for
anibody were prepared in PBST containing 0.05 per
cent (wt/vol) gelatin (PBSTG) and 0.1 ml of each
dilution was added to a microtitre plate well. After
incubation for 1 h at 37"C, wells were washed five
times with PBST, and 0-1ml of goat anti-mouse
IgG, IgA or IgM was added to the wells. Following
incubation for 1 h at 37"C, the wells were washed
and rabbit anti-goat IgG alkaline phosphatase conjugate was added to each well and incubated at 37°C.
The wells were then washed as described above and
the enzymatic activity in each well determined with
p-nitrophenyl phosphate disodium, as described
above. As controls, firstly some of the microtitre
wells were coated with bovine serum albumin
instead of toxin, secondly PBSTG was added to
some wells in place of antibody samples, and thirdly
samples of known antitoxin titre were diluted and
added to several of the wells. Net absorbance was
calculated by subtraction of the mean absorbance in
wells coated with bovine serum albumin from the
mean absorbance in wells coated with toxin.
Cytotoxicity Neutralisation Assay
Biological specimens prepared from hamsters
(i.e., sera, maternal milk, and intestinal homogenates) were examined for their ability to neutralise
the cytotoxic activity of purified preparations of
toxins A and B in a HeLa tissue culture cell assay.
Serial dilutions of the biological specimens were
prepared in PBS, and each dilution mixed with an
equal volume of toxins A or B. The toxins were
diluted to concentrations 8-fold greater than the
highest dilution of each toxin that caused complete
rounding of 100 per cent of the HeLa cells in a
51
ANTIBODIES TO C. DIFFICILE TOXINS A AND B
Table 1 . Summary of the specificity and sensitivity of anti-mouse immunoglobulins in ELISA
P Valuef
Anti-mouse5
IgG (Pool)
Mouse IgG
16
Anti-mouse
IgA
Mouse IgA
32
Anti-mouse
IgM
MouseIgM
32
Anti-mouse
IgG (Pool)
Hamster IgG
Anti-mouse
IgA
Hamster IgA
Anti-mouse
IgM
Hamster IgM
250
63
63
IgG/IgG:
IgA/ IgG :
IgM/IgG:
IgG/IgA:
IgA/IgA:
IgM/IgA:
IgG/IgM:
IgA/IgM:
IgM/IgM:
IgG/IgG:
IgA/IgC:
IgM/IgC:
IgG/IgA:
IgA/IgA:
IgM/IgA:
IgG/IgM:
IgA/IgM:
IgM/IgM:
lOO0/0
8 Yo
8O h
4 yo
loooh
13%
8%
6%
100%
100%
19%
18%
6 Yo
100%
15%
10%
22 Yo
< 0.001
<0.002
< 0.001
< 0.001
<0.002
< 0.001
< 0.001
< 0.002
< 0.001
< 0.002
<0.001
< 0.00 1
100%
*Sensitivity was determined by examining serial two-fold dilutions of each immunoglobulin by ELISA. The
lowest concentration of immunoglobulins which gave an OD,,o significantly greater than background was
defined as the sensitivity of the ELISA.
?Specificity is defined as the A,,, value of test immunoglobulin/A,,, value of reference immunoglobulin
(noted in bold print) x 100.
$Student’s t-test.
$Goat anti-mouse immunoglobulins were prepared as described in Results.
microtitre well (TCD,,,). The TCD,,, of the their infants. Since anti-hamster class-specific
partially purified toxin A and B preparations were immunoglobulins are not commercially available
190 ng of protein (38.7 ng/ml) and 4.5 pg of protein we decided to substitute anti-mouse class-specific
(90.4 pg/ml), respectively. After incubation at 37°C immunoglobulins for anti-hamster class-specific
for 1 h, 50 p1 of each mixture were added to a micro- immunoglobulins in the ELISAs.
titre well containing a monolayer of HeLa cells
In the initial investigations, mouse immunogloand 200 p1 of cell culture medium. The HeLa cells bulins were characterised by ELISA. Optimum
concentrations of goat anti-mouse classwere prepared as described p r e v i o ~ s l y .The
~ ~ ~ working
~~
antitoxin titre was defined as the reciprocal of the specific immunoglobulins for use in ELISA were
greatest dilution which completely inhibited round- determined by checkerboard titration. Pooled
ing of the HeLa cells after 24 h incubation at 37°C. anti-mouse IgG was prepared by mixing equal
The differences between experimental groups were volumes of anti-mouse IgGl (diluted 1:3200 in
PBSTG), IgG2a (1 :1600),IgG2b (1 :1600),and IgG3
analysed by Student’s t test (P-values).
(1:3200). Goat anti-mouse IgA and IgM were
1:12 800 in PBSTG. These dilutions of goat
diluted
RESULTS
anti-mouse immunoglobulins reacted specifically
Characterisation of Mouse and Hamster Immunoglo- against 50 ng of the respective mouse immunoglobulins
bulins in ELISA (Table 1).
Hamster immunoglobulins were next examined
ELISAs were developed to detect hamster classspecific antibodies to toxins A and B in various for reactivity with goat anti-mouse immunoglobubiological specimens obtained from hamsters lins (Table 1). Goat anti-mouse IgG was prepared
immunised parenterally with toxoids A and B and by mixing equal volumes of anti-mouse IgGl
52
P.-H. KIM AND R. D. ROLFE
1.5
-.
1.5
h
-
h
5 1.0 -
1.0-
*
0
0
w
z
z
0
.
P
U
a
c1
Lli
.
C
0.5
-
$
CI
0.5
-
a
<
-2
0.0
0
I
I
1
2
,
,
,
,
3
4
5
6
7
Serum Dilution (log2)
I
,
,
,
8
9
10
0.0
0
1
1
I
1
2
3
I
I
I
I
I
5
6
7
Serum Dilution (log2)
8
9
10
4
1
I
Figure 1. Specificity of ELISAs for IgG antibody to Clostridium dijicile toxins A and B. Sera samples were obtained from hamsters
imrnunised against toxin A, toxin B or NTBF. Each serum sample was diluted 1 :625 in PBSTG. Sera from hamsters immunised against
toxin A ( 0 ) toxin
;
B (A);NTBF (0)
(diluted 1300 in PBSTG), IgG2a (1 :400), IgG2b (1 :
400), and IgG3 (1:800). Goat anti-mouse IgA and
IgM were diluted 1 :3200 and 1:6400, respectively.
Similar to the observations made with mouse immunoglobulins, the above dilutions of goat anti-mouse
immunoglobulins reacted specifically against 50 ng
of the respective hamster immunoglobulins in
ELISA. However, overall sensitivity of goat antimouse immunoglobulins in detecting hamster
immunoglobulins by ELISA was at least two times
lower than for mouse immunoglobulins.
Specijicity of ELISAs f o r Characterisation of Antibody to Toxins A and B
Serum samples were obtained from hamsters
immunised against toxins A, B, or the NTBF. Sera
collected from hamsters immunised against toxins
A or B neutralised the cytotoxic activity of each
respective toxin; no cross-neutralisation was
demonstrated. Sera collected from hamsters
immunised against the NTBF did not neutralise the
cytotoxic activity of either toxins A or B. These
sera were tested in the two ELISAs developed for
measuring class-specificantibodies to toxins A or B.
No significant cross-reaction occurred between the
two ELISAs for detection of IgG antibody to toxins
A and B (Figure I). Sera collected from hamsters
immunised against toxin A reacted in the ELISA for
detection of IgG antibody to toxin A but not in the
ELISA for detection of IgG antibody to toxin B.
Similarly, sera collected from hamsters immunised
against toxin B reacted in the ELISA for detection
of IgG antibody to toxin B but not in the ELISA for
detection of IgG antibody to toxin A. Similar results
were obtained in the detection of IgA and IgM antibodies to these toxins.
Characterisation of Class-SpeciJicImmunoglobulins
in Adult Hamsters Immunised Parenterally against
Toxins A and B
Specimens collected from adult hamsters immunised parenterally against toxoid AB or the NTBF
were characterised for class-specific antibodies to
toxins A and B. The sera from hamsters immunised
against toxins A and B possessed cytotoxicity neutralisation titres of 25 against toxin A and 625
against toxin B. The sera from hamsters immunised
against the NTBF did not neutralise the cytotoxicity
of either toxins A or B. Figure 2 shows that levels of
IgG and IgM antibodies to both toxins predominated in sera although low but significant
(p<O.OOl) levels of IgA antibody could also be
demonstrated in the sera.
The ileum and caecum from immunised adult
hamsters were also examined for class-specificantibodies to toxins A and B. Significant levels of
specific IgG antibodies to toxins A and B could be
detected in both caecal tissue and contents (Figure
3). However, none of the ileal or caecal specimens
contained antibody which neutralised the cytotoxic
activity of toxins A or B. We have previously
demonstrated that ELISAs are more sensitive than
cytotoxicity neutralisation in detecting antibodies
to toxins A and B.17 Specific IgA and IgM anti-
53
ANTIBODIES TO C. DIFFICLE TOXINS A AND B
2.5
1
2.0
h
5
s
s
1.5
ai
::
2
1.0
0
2
0.5
0.0
IgG
IgM
IgA
Figure 2. Characterisation of class-specificimmunoglobulins in serum of
hamsters immunised against toxins A and B or NTBF. Serum was
diluted 1:25 in PBSTG. Antibody to toxin A (D); Antibody to toxin B
(0);NTBF sera reactivity in ELISAs (El). Data are the means+SEM
(vertical bars) of four samples
Caecal Tissue
1.2
Caecal Contents
1
l.01
1.2
T
e
1 .o
0.8
3
0
s
a,
0.6
E
a
0.4
a
d
0.2
0.0
IgG
IgA
IgM
IgC
IgM
Figure 3. Class-specificantibodies to toxins A and Bin the caecum of adult hamsters immunised against toxins A and B or NTBF. For
the preparation of caecal contents, the mucous lining of the caecum was added to the luminal contents. The samples were then diluted
five times (w/v) in PBSTG, centrifuged and the supernatant filter sterilised. The intestinal tissue samples were diluted five times (w/v) in
PBSTG, homogenised, centrifuged and the supernatant filter sterilised. Antibody to toxin A (El); Antibody to toxin B (0);NTBF sera
reactivity in ELISAs (a).Data are the meansf SEM (vertical bars) of four samples
bodies in caecal tissue and contents of hamsters
immunised against toxins A and B were not significantly greater than in hamsters immunised against
the NTBF. Similar results were obtained with ileal
tissue and contents obtained from immunised adult
hamsters (data not shown).
IgG was also the predominate class-specific
immunoglobulin to toxins A and B in the milk from
hamsters immunised with toxoids A and B; no significant levels of IgA or IgM were present in the milk
of these hamsters (Figure 4). The milk from hamsters immunised against toxins A and B possessed
cytotoxicity neutralisation titres of 1:5 against toxin
A and 1:125 against toxin B. Milk from hamsters
immunised against the NTBF did not neutralise the
cytotoxic activity of either toxins A or B. Milk
samples from hamsters immunised with toxoid A
and non-immunised hamsters were characterised
for class-specificimmunoglobulins (Figure 5). Comparable levels of non-specific IgG and IgA were
54
P.-H. KIM AND R. D. ROLFE
present in the milk from hamsters immunised
against toxoid A, while IgA was the predominant
immunoglobulin in non-immune milk. However,
none of these differences in immunoglobulin concentrations in immune and non-immune milk were
statistically significant ( p > 0.05) although this
could be related to the small population size (i.e.,
duplicate samples).
2'01
T
--
Characterisation of Class-Specific Immunoglobulins
to Toxins A and B in Infant Hamsters
The intestines and sera of 7 d-old infant hamsters
from mothers immunised parenterally against
toxins A and B when examined for class-specific
antibodies to these toxins yielded results similar to
those seen in maternal milk (Figure 6 ) with only
significantlevels of IgG antibodies to toxins A and B
detected. Levels of class-specificantibodies to toxins
A and B were also measured in the caeca and sera of
infant hamsters of different ages from mothers
immunised parenterally against toxins A and B
(Figure 7). Significant levels of IgG antibodies to
these toxins were present in the caeca of infants up
to 10 d old, whereas significant levels of IgG antibodies to toxins A and B could be demonstrated in
the sera up to approximately 24 d of age. Significant
levels of IgA and IgM could not be demonstrated in
any of the hamsters (data not shown).
DISCUSSION
The ability of animals to produce toxin-neutralising
Ik!G
IgA
I%M
antibodies may not only influence the severity of C.
Figure 4. Characterisation of class-specific immunoglobulins in dificile-associated disease, but also give protection
milk from hamsters immunised against toxins A and B or NTBF. against further infection with C. dificile. The presMilk samples were collected 7 d post-partum, centrifuged, the
supernatant filter sterilised, and the filtrate diluted 1:25 in ence of toxin-neutralising antibodies could also
PBSTG. Antibody to toxin A (a);
Antibody to toxin B (0); account for asymptomatic colonisation of adults
NTBF sera reactivity in ELISAs (@). Data are the means+ SEM and infants by toxigenic C. dificile as well as the
(vertical bars) of four samples
failure of C. dificile to cause disease throughout the
colon. Studies have demonstrated that antibodies to
toxins A and B are present in the majority of older
infants and adults and, furthermore, that patients
with C. dificile-associated intestinal disease develop
serologic responses to one or both
In this investigation, the hamster model of clindamycin-induced C. dificile-associated ileocaecitis
was used to study the role of antibodies to toxins A
and B in protection against disease. ELISAs were
-I
developed to permit detection of hamster classspecific immunoglobulins directed against C. dzficile toxins A and B. Initial attempts were made to
purify heavy chains of hamster class-specific immunoglobulins which were to be used in rabbit immunisation for obtaining anti-hamster class-specific
immunoglobulins for use in ELISAs. However, it
was not possible to obtain sufficient quantities of
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Absorbance (410 nm)
purified hamster immunoglobulin heavy chains for
Figure 5. Comparison of non-specific immunoglobulin levels in rabbit immunisation. Consequently, anti-mouse
non-immune and immune milk. Milk samples were collected 7 d immunoglobulins were adopted for use in characpost-partum, centrifuged and the supernatants filter sterilised.
terising hamster antibodies to toxins A and B.
Samples were diluted 1 :15625 before being used to coat the wells
Hamster immunoglobulins have not been as well
of microtitre plates. Immune milk (8);
Non-immune milk (FA).
characterised as mouse and human immunoglobuData are the means of duplicate samples
55
ANTIBODIES TO C. DIFFICLE TOXINS A AND B
Infant Intestine
2.5
-E
2
-?
2.0
2.0
h
2
s
1.5
E
Y
"e
2k
W
s:
3
Infant Serum
2.5
1.5
W
x
2L
1.0
1.0
A
.
2
0.5
0.5
0.0
0.0
IgG
IgA
IgM
IgA
IgM
Figure 6. Characterisation of class-specificimmunoglobulins to toxins A and B in the intestine and serum of 7 d-old infant hamsters
immunised against toxins A and B or NTBF. Intestinal samples (large and small intestine) were collected, diluted 5 times (w/v) in
PBSTG, centrifuged, and the supernatant filter sterilised. Serum samples were diluted 1:25 in PBSTG. Antibody to toxin A (a);
Antobody to toxin B (0);NTBF sera reactivity in ELISAs (El). Data are the means SEM (vertical bars) of four samples
+
Intestine
10
17
24
31
38
Age (days)
Serum
48
Control
10
17
24
31
Age (days)
38
48
Control
Figure 7. IgG antibody to toxins A and B in the caecum and serum of infant hamsters of different ages. Mother hamsters were
immunised against toxins A and B. Caecal samples were diluted five times (w/v) in PBSTG, homogenised, centrifuged, and the
supernatant filter sterilised. Each serum sample was diluted 1:25 in PBSTG. Control caecal homogenates and sera were obtained from
7 d-old infants from non-immunised mothers. Antibody to toxin A (El); Antibody to toxin B (El).Data are the means+ SEM (vertical
bars) of three samples
lins. Hamster immunoglobulins are composed of
IgA, IgM and two subclasses of IgG (GI, G2) while
mouse immunoglobulins are composed of IgA, IgM
and three subclasses of IgG (Gl, G2a, G2b, G3).'s8
There is, however, evidence that hamster IgG2 is
heterogeneous, indicating that at least two types of
IgG2 exist. The exact evolutionary relationship
between the hamster and mouse is unknown. Simpson3' places the divergence point somewhere in the
late Miocene or early Pliocene period, approximately 20 million years ago. There is indirect evidence that mouse and hamster immunoglobulins
are closely related. McGuire et a1.22compared hamster, mouse and human constant regions of IgM
heavy chain (Cp) genes for phylogenetic conservation. Homologies with the hamster gene for the Cp
5' region are 8 1 per cent, 59 per cent, and 5 1 per cent
for mouse, rabbit, and human, respectively, and the
corresponding homologies for the Cp 3'region are 94
per cent, 83 per cent, and 81 per cent, respectively.
Cross-reactivity of goat anti-mouse IgG (heavy
chain specific) against hamster IgG was first examined in double immunodiffusion agar plates and a
single precipitation line was observed (data not
56
shown). This promising finding prompted further
analyses of the specific cross-reactions between
anti-mouse immunoglobulins and hamster immunoglobulins by ELISA, which is a much more
sensitive method than Ouchterlony double immunodiffusion. Overall sensitivity of anti-mouse
immunoglobulins for detecting hamster immunoglobulins was lower than the sensitivity for detecting
mouse immunoglobulins (Table 1). This could be
due to a fewer number of antigenic sites on hamster
immunoglobulins as compared to mouse immunoglobulins and/or a decreased affinity of the antibodies for the antigenic sites. However, overall
specificity of the ELISAs for detection of hamster
immunoglobulins was comparable to the specificity
for mouse immunoglobulins. In addition, the
ELISA for antibody to toxin A and the ELISA for
antibody to toxin B were specific for each class of
immunoglobulin (Figure I). Although the ELISAs
using anti-mouse class-specific immunoglobulins
permitted the detection of hamster class-specific
antibodies, it would be advantageous to eventually
use anti-hamster class-specific immunoglobulins
because of possible increases in sensitivity and
specificity.
In earlier studies we demonstrated that adult
hamsters immunised parenterally against only toxin
A or both toxins A and B were protected against
antibiotic-induced C. dzficile-associated ileocaecitis
while hamsters immunised against only toxin B or a
broth filtrate from a non-toxigenic strain of C. difJicile were not protected against disease. l 7 The protection observed in these investigations presumably
reflected the inactivation of the biological activity of
C. difJicile toxins before they could induce pathological damage in the intestinal tract.
In the present investigation, parenteral immunisation of adult hamsters with toxoids A or B elicited
toxin-specific IgG, IgA and IgM immunoglobulins
in hamster sera. As shown in Figure 2, hamster
class-specific antibody profiles to toxins A and B
were almost identical, indicating that the immunisation protocols induced antibodies to both toxins
with equal efficacy. Interestingly, significant levels
of IgM antibody to toxins A and B were present in
the sera even though antibody analyses were performed 14 weeks after the start of immunisation.
Parenteral immunisation did not induce a toxinspecific mucosal IgA immune response since the
ileal and caecal tissue contained only toxin-specific
IgG (Figure 3). These results were unexpected since
previous investigators have reported that parenteral
immunisation with Vibrio cholerae and Shigella
P.-H. KIM AND R. D. ROLFE
jexneri elicits a specific IgA as well as IgG response
in the mucosal secretions of rabbit^.'^.^^ Furthermore, it is well established that antibodies produced locally in the intestine, particularly sIgA,
are important for specific immune protection
against many enteric infections. Nonetheless,
serum-derived IgG antibody protected adult hamsters against C. dificile-associated ileocaecitis. Since
only very low levels of toxin-specificantibodies were
detected in the luminal contents of the ileum and
caecum it appears that toxin specific IgG acts at the
level of the mucosa and/or submucosa.
As described previously, maternal protection
against C. dificile-associated ileocaecitis can be
transferred to offspring through milk.” Therefore,
it was of interest to identify the class-specific antibodies in milk which may account for the protection
of infant hamsters against disease. Similar to the
observations made with adult intestines, IgG was
the predominate class-specific antibody to toxins A
and B in the milk from hamsters immunised with
toxoids A and B; once again, no significant levels of
IgA or IgM antibodies were present (Figure 4).
Theodore et ai.34 also demonstrated that only
specific IgG antibody is present in the colostrum
and milk of rabbits immunised intravenously with
respiratory syncytial virus. The analyses of total
non-specific immunoglobulins in the milk of
non-immunised hamsters showed that IgA was the
predominant immunoglobulin (Figure 5). Similar
observations have been made in the colostrum of
non-immunised hamsters. On the other hand,
comparable levels of non-specific IgA and IgG
were present in milk from hamsters immunised with
toxoid A. These results suggest that toxin-specific
IgG antibody and/or antibody-producing cells are
transferred from serum to milk. At least two factors
may contribute to the high levels of IgG in maternal
milk of immunised hamsters. First, the half-life of
IgG is generally longer than the half-life of IgA and
IgM. The catabolic half-life of IgG, IgM, and IgA
are 23, 5, and 6 days in humans and 4, 0.5, and 1.3
days in mice, respectively. Second, toxin-specific
IgM and IgA may be degraded during lactation.
Ogra and Ogra2’ have reported that the levels of
IgM and IgA in human colostrum and milk fall
rapidly after the first three to four days, whereas IgG
remains at a constant low level. This may explain
why no toxin-specific IgM is detected in hamster
maternal milk although a significant level of toxinspecific IgM antibody is present in the sera.
The class-specificantibodies to toxins A and B in
infant intestine and sera were similar to the anti-
57
ANTIBODIES TO C. DIFFICILE TOXINS A AND B
bodies present in adult intestine and milk (Figure 6).
This indicates that toxin specific IgG present in
maternal milk is absorbed across the intestinal tract
of infant hamsters into the systemic circulation.
Other investigators have also reported the intestinal
absorption of IgG in infant
The levels
of IgG in infant sera were higher and lasted longer
than infant caeca, indicating that IgG antibody in
serum is more stable. We have previously shown
that infant hamsters from mothers immunised parenterally against toxin A are partially protected
against C. dijicile-associated ileocaecitis up to 17
days of age." This suggests that toxin-specific IgG
in infant serum piays an important protective role
against C. dificile-associated ileocaecitis since
toxin-specific IgG in infant caeca was not present
at significant levels after ten days of age while
infant sera possessed toxin-specific IgG up to
approximately 24 days of age (Figure 7).
The mechanisms by which serum IgG antibody to
toxins A and B protects adult and infant hamsters
against C. dijicile-associated intestinal disease are
not known and merit further investigation. However, one possible mechanism is inhibition of an
inflammatory response in the intestine. Triadafilopoulos et al.36 reported that toxin A causes severe
epithelial cell necrosis with destruction of villi and
polymorphonuclear infiltration. They speculated
that inflammatory mediators released by neutrophils, mast cells, and/or macrophages are likely to
contribute to the intestinal damage seen in C. dificile-associated pseudomembranous colitis and diarrhoea. Recently, Daubener et aL9 demonstrated
that C. dificile toxins inhibit T cell proliferation by
influencing monocytes in a dose-dependent fashion.
They proposed that the influence of C. dificile
toxins on monocytes may be an additional factor in
the development of pseudomembranous colitis. One
can postulate from the data in our investigation that
circulating antibody to C. dijicile toxins gains
access to intestinal tissue which in turn neutralises
the toxins before they can bind to specific receptors
and induce harmful inflammation in the intestine.
In conclusion, the induction of C. dificileassociated intestinal disease clearly involves
many parameters including the physiological and
immunological status of the host. The hamster is
an effective model to study the pathogenesis of C.
dificile-associated intestinal disease since enteric
colonisation with C. dijicile is possible, a vigorous
immune response occurs, and resistance to subsequent re-infection has been demonstrated. It
would be hazardous to conclude that the patho-
genesis of C. dificile and the possible role of immunological factors in protection are the same in
humans as in hamsters. However, the similarities
between C. dificile-associated disease in hamsters
and humans suggests that the results obtained
with the hamster model will directly aid in our
understanding of the pathogenesis of C. dijicileassociated disease in humans.
ACKNOWLEDGEMENTS
This work was supported, in part, by Public Health
Service grant ROI-A1121489 from the National Institute
of Allergy and Infectious Disease. We thank Woosun
Song for excellent technical assistance during the course
of these investigations.
REFERENCES
1. Allo M, Silva J, Fekety R, Rifkin GD, Waskin H.
2.
3.
4.
5.
6.
7.
8.
9.
10.
(1979). Prevention of clindamycin-induced colitis
in hamsters by Clostridium sordellii antitoxin.
Gastroenterology 76,351-355.
Aronsson B, Granstrom M. (1988). Immunological
response to Clostridium dijicile infection. In: Rolfe
RD, Finegold SM (ed) Clostridium difficile: Its Role
in Intestinal Disease. Academic Press, New York, pp
99-1 12.
Aronsson B, Granstrom M, Molby R, Nord CE.
(1983). Enzyme-linked immunosorbent assay
(ELISA)for antibodiesto Clostridium dzficile toxins
in patients with pseudomembranous colitis and
antibiotic-associated diarrhea. Journal of Immunological Methods 60,341-350.
Aronsson B, Molby R, Nord CE. (1984). Diagnosis
and epidemiology of Clostridium dificile enterocolitis in Sweden. Journal of Antimicrobial Chemotherapy 14 (Suppl. D), 85-95.
Bienenstock J. (1970). Immunoglobulins of the
hamster. 11. Characterization of the yA and other
immunoglobulins in serum and secretions. Journal
of Immunology 104,1228-1235.
Bradford MA. (1976). A rapid and sensitive method
for quantitation of microgram quantities of protein
utilizing the principle of protein dye binding. Analytical Biochemistry 72,248-254.
Coe JE. (1968). The immune response in the hamsters. I. Definition of the two 7s globulin classes.
Journal of Immunology 100,507-5 15.
Coe JE, Bell JF. (1977). Antibody response to rabies
virus in Syrian hamsters. Infection and Immunity 16,
9 15-9 19.
Daubener W, Leiser E, Eichel-StreiberC, Hadding
U. (1988). Clostridium dificile toxins A and B inhibit
human immune response in vitro. Infection and
Immunity 56, 1107-1 112.
Davis BJ. (1965). Disc electrophoresis. 11. Methods
58
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
P.-H. KIM AND R. D. ROLFE
and application to human serum proteins. Annals of
the New York Academy of Sciences 121,404427.
Donta ST. (1988). Mechanism of action of Clostridium dificile toxins. In: Rolfe RD, Finegold SM
(eds) Clostridium difficile: Its Role in Intestinal
Disease. Academic Press, New York, pp 169-181.
Escribano MJ, Haddada H, DeVaux Saint Cyr C
(1982). Isolation of two immunoglobulin G subclasses, IgG2 and IgGl, from hamster serum protein-A-Sepharose. Journal of Immunological
Methods 52,63-72.
Fainstein V, Bodey GP, Fekety R. (I98 1). Relapsing
pseudomembranous colitis associated with cancer
chemotherapy. Journal of Infectious Diseases 143,
863-865.
George WL. (1988). Antimicrobial agent-associated
diarrhoea in adult humans. In: Rolfe RD, Finegold
SM (eds) Clostridium difficle: Its Role in Intestinal
Disease. Academic Press, New York, pp 3 1 4 .
Hasegawa H, Nakamura A, Watanabe K, Brown
WR, Nagura H. (1987). Intestinal uptake of IgG in
suckling rats: Distinction between jejunal and ileal
epithelial cells demonstrated by simultaneous
ultrastructural localization of IgG and acid phosphatase. Gastroenterology 92,186191.
Keren VF, McDonald RA, Carey JL. (1983). Effect
of parenteral immunization on the local immunoglobulin A response of the intestine to ShigellaJlexneri antigens. Infection and Immunity 42,202-207.
Kim P-H, Iaconis JP, Rolfe RD. (1987). Immunization of adult hamsters against Clostridium dificileassociated ileocecitis and transfer of protection to
infant hamsters. Infection and Immunity 55, 29842992.
Kim P-H, Rolfe RD. (1988). Characteristics of protective antibodies to Clostridium dij?cile toxins A
and B. Abstracts of the Annual Meeting of the
American Society for Microbiology, Abstract E24.
Libby JM, Jortner BS, Wilkins TD. (1982). Effects
of the two toxins of Clostridium dijicile in antibioticassociated cecitis in hamsters. Infection and Immunity 36,822-829.
Lyerly DM, Sullivan NM, Wilkins TD. (1983).
Enzyme-linked immunosorbent assay for Clostridium dij?cile. Journal of Clinical Microbiology 17,
72-78.
Lyerly DM, Wilkins TD. (1988). Purification and
properties of toxins A and B of Clostridium dij?cile.
In: Rolfe RD, Finegold SM (eds) Clostridium difficile: Its Role in Intestinal Disease. Academic Press,
New York, pp 145-167.
McGuire KL, Duncan WR, Tucker PW. (1985).
Phylogenetic conservation of immunoglobulin
heavy chains: direct comparison of hamster and
mouse Cp genes. Nucleic Acids Research 13, 561 15638.
Mitchell TJ, Ketley JM, Halan SC, Stephen J,
Burdon DW, Candy CC, Daniel R. (1986). Effects of
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
31.
toxins A and B of Clostridium dificile on rabbit
ileum and colon. Gut 27,78-85.
Moskovitz M, Bartlett JG. (1981). Recurrent
pseudomembranous colitis unassociated with prior
antibiotic therapy. Archives of Internal Medicine
141,663464.
Ogra SS, Ogra PL. (1978). Immunologic aspects of
human colostrum and milk. I. Distribution characteristics and concentrations of immunoglobulins at
different times after the onset of lactation. Journalof
Pediatrics 92,546549.
Onderdonk AB. (1988). Role of the hamster model
of antibiotic-associated colitis in defining the etiology of the disease. In: Rolfe RD, Finegold SM
(eds) Clostridium difficile: Its Role in Intestinal Disease. Academic Press, New York, pp 115-127.
Peri BA, Rothberg RM. (1986). Transmission of
maternal antibody prenatally and from milk into
serum of neonatal rabbits. Immunology 57,49-53.
Rolfe RD, Finegold SM. (1979). Purification and
characterization of Clostridium dificile toxin. Infection and Immunity 25,191-201.
Rolfe RD, Iaconis JP. (1983). Intestinal colonization of infant hamsters with Clostridium dificile.
Infection and Immunity 42,480-486.
Simpson GG. (1959). Cold Spring Harbor,
Symposium on Quantitative Biology 24,255-271.
Sullivan NM, Pellet S, Wilkins TD. (1982). Purification and characterization of toxins A and B of
Clostridium di8cile. Infection and Immunity 35,
1032-1040.
Svennerholm A, Holmgren J. (1 977). Immunoglobulin and specific-antibody synthesis in vitro by
enteral and nonenteral lymphoid tissues after subcutaneous cholera immunization. Infection and
Immunity 15,36&369.
Taylor NS, Thorne GM, Bartlett JG. (1981).
Comparison of two toxins produced by Clostridium
dificile. Infection and Immunity 34,10361043.
Theodore CM, Losonsky G, Peri B, Fishaut M,
Rothberg RM, Ogra PL. (1982). Immunologic
aspects ofcolostrum and milk: development of antibody response to respiratory syncytial virus and
bovine serum albumin and rabbit mammary glands.
In: Handon LA, Sell KW. (eds) Recent Advances in
Mucosal Immunity. Raven Press, New York, pp
393403.
Towbin H, Staehelin T, Gordon J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proceeding of the National Academy of Sciences USA 76,
4650-4654.
Triadafilopoulos G, Pothoulakis C, O’Brien MJ,
LaMont JT. (1987). Differential effects of Clostridium dijicile toxins A and B on rabbit ileum.
Gastroenterology 93,273-279.
Trnka VM, LaMont JT. (1981). Association of
Clostridium di8cile toxin with symptomatic relapse
ANTIBODIES TO C. DIFFICILE TOXINS A AND B
of chronic inflammatory bowel disease. Gastroenterology 80,693-696.
38. Visicidi R, Laughon BE, Yolken R, Bo-Linn P,
Moench T, Ryder PW, Bartlett JG. (1983). Serum
antibody response to toxins A and B of Clostridium
diflciie. Journal of Infectious Diseases 148,93-100.
39. Wexler H. (1988). Other potential diagnostic
techniques. In: Rolfe RD, Finegold SM (eds)
59
Clostridium difficile: Its Role in Intestinal Disease.
Academic Press, New York, pp 309-338.
40. Wunderly CH. (1960). The technique of immunoelectrophoresis in agar gel. In: Alexander P, Block AJ
(eds) A Laboratory Munuaf of Analytical Methods of
Protein Chemistry, Vol. 2. Pergamon Press, New
York, pp 143-167.