Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Production of Monoclonal Antibodies Directed against
the Microsporidium
Enterocytozoon bieneusi
ISABELLE ACCOCEBERRY,* MARC THELLIER, ISABELLE DESPORTES-LIVAGE,
ABDERRAHIM ACHBAROU, SYLVESTRE BILIGUI, MARTIN DANIS,
ANDANNICK DATRY
Unite´ INSERM 511, Laboratoire de Parasitologie-Mycologie, Centre Hospitalier-Universitaire
de la Pitie´-Salpeˆtrie`re, 75013 Paris, France
Received 24 March 1999/Returned for modification 24 June 1999/Accepted 7 September 1999
Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT)
were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against
whole spores (protocol 1) or chitinase-treated spores (protocol 2) of
Enterocytozoon bieneusi
and were cloned
twice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as
immu-noglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture.
IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of
E. bieneusi
,
strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electron
microscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores of
the other intestinal microsporidium species
Encephalitozoon intestinalis
or with yeast cells, bacteria, or any
other intestinal parasites, was observed. The MAbs were used to identify
E. bieneusi
spores in fecal specimens
from patients suspected of having intestinal microsporidiosis. The IFAT was validated against standard
staining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description and
characterization of two MAbs specific for the spore wall of
E. bieneusi
. These MAbs have great potential for the
demonstration and species determination of
E. bieneusi
, and their application in immunofluorescence
identi-fication of
E. bieneusi
in stool samples could offer a new diagnostic tool for clinical laboratories.
Microsporidia are obligate intracellular protistan parasites
that infect a variety of cells from a wide range of invertebrate
and vertebrate hosts. Also identified in humans, more
espe-cially in immunocompromised patients, microsporidia appear
to be major opportunistic pathogens. These parasites were
shown to be the prevalent cause of intestinal infections
re-ported in patients with AIDS and diarrhea (17, 21) in
indus-trialized countries. Significantly, the introduction of
antiretro-viral combination regimens including human immunodeficiency
virus (HIV) protease inhibitors has resulted in a decrease in
the number of cases of AIDS-related microsporidiosis (10, 12).
The first documented case of intestinal infection was caused
by
Enterocytozoon bieneusi
(7), the microsporidian species
most commonly found in humans. This parasite is usually
ob-served in HIV-infected patients with CD4 lymphocyte counts
of less than 50 cells/mm
3who complain of chronic diarrhea,
nausea, malabsorption, and severe weight loss (4, 24).
Enceph-alitozoon intestinalis
(14) also causes intestinal infections
fre-quently associated with nephritis, sinusitis, or bronchitis (17,
21). These parasites are also pathogenic in subjects with
im-munodeficiency due to causes other than AIDS. Cases of
in-testinal microsporidiosis have been detected in organ
trans-plant recipients (25, 28). The two species
E. bieneusi
and
E.
intestinalis
also appear to be responsible for cases of diarrhea
in immunocompetent subjects (11). Most of them are
pre-sented by travellers returning from tropical areas (26, 27, 29,
34). Less expected is the increasing number of
HIV-seroneg-ative and asymptomatic individuals found to be infected with
microsporidia (8, 15, 32).
Over the past 10 years, different diagnosis methods, based
on the detection of the parasites’ spores in stools and other
biological samples, have been proposed (3, 16, 31, 39).
Al-though PCR appears to be the most sensitive method (6, 9, 11,
23), immunological tools remain helpful for diagnosis and for
epidemiological survey or experimental investigation. Specific
monoclonal antibodies (MAbs) against
E. intestinalis
isolates
easily obtained through in vitro systems have been produced
(5). To date, such systems are still lacking for
E. bieneusi
.
However, spores of this species extracted from fecal samples
enabled the production of the MAbs described in the present
study.
MATERIALS AND METHODS
Sources of parasites. (i)E. bieneusi.In the absence of an in vitro cultivation model and due to the invasive procedures needed for collecting epithelium or fluid samples from the gastrointestinal tract, human stools were the source ofE. bieneusi spores. Fecal specimens were obtained from HIV-infected patients. Microsporidian spores were detected by fluorochrome Uvitex 2B stain (31) and Weber’s chromotrope-based modified trichrome stain (16). Fecal samples con-taining numerous small oval spores were homogenized and suspended in a solution of phosphate-buffered saline (PBS; Sigma Laboratories, Saint-Quentin-Fallavier, France). The samples were processed for transmission electron mi-croscopy (TEM) and tested by PCR to confirm the identification of the species and to exclude a concomitantE. intestinalisinfection.
(ii) TEM.The fecal samples were fixed at room temperature in 2.5% glutar-aldehyde in 0.1 M Na cacodylate buffer (pH 7.2) for 60 min, rinsed in buffer, and then postfixed in ferriosmium [1% (wt/vol) OsO4and K3Fe(Cn)6in cacodylate
buffer] for 60 min. After ethanolic dehydration, the samples were embedded in Spurr resin. Thin sections, stained with uranyl acetate and lead citrate, were examined with a JEOL JEM 100 CX transmission electron microscope.
(iii) PCR amplification.The PCR assay was performed as described previously by Ombrouck et al. (23). The primers V1 (5⬘-CACCAGGTTGATTCTGCCTG AC-3⬘) and EB450 (5⬘-ACTCAGGTGTTATACTCACGTC-3⬘) described by Zhu et al. (38) were used to amplifyE. bieneusiDNA. The primers V1 and SI500 (5⬘-CTCGCTCCTTTACACTCGAA-3⬘) described by Weiss et al. (37) were used to amplifyE. intestinalisDNA.
(iv)E. intestinalis.Spores were obtained from cultures in rabbit kidney cells (RK13), as described by van Gool et al. (30). Parasite spores were harvested weekly, counted in a hemocytometer, resuspended in PBS, and stored at 4°C until used.
* Corresponding author. Mailing address: Laboratoire de
Parasi-tologie-Mycologie, CHU de Bordeaux, 1, rue Jean Burguet, 33000
Bordeaux, France. Phone: 33 5-56 79 58 37. Fax: 33 5-56 79 58 79.
E-mail: bernard.couprie@chu-aquitaine.fr.
4107
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Antigen preparation procedures. (i) Spore concentration.The stool suspen-sion was filtered through a graded series of six nylon sieves (pore diameters were 100, 50, 30, 20, 10, and 5m, respectively). The filtration was facilitated by the addition of 1,000 to 2,000 ml of PBS. The final filtrate was centrifuged at 500⫻ gfor 6 min to eliminate large particles, and the sieved spores in the supernatant were pelleted by centrifugation at 2,500⫻gfor 20 min. The pellet was resus-pended in PBS (1/3 [vol/vol]).
(ii) Spore purification.Density gradient centrifugation was performed with various concentrations of Percoll (19). The discontinuous gradient consisted of 10 ml of stock isotonic Percoll solution (prepared by mixing 10 ml of 10-fold-concentrated PBS and 90 ml of Percoll [Sigma Laboratories] to yield a pH of 7.4 and an osmolarity of 335 mosM), 10 ml of 67.5% stock Percoll diluted with PBS, 10 ml of 45% stock Percoll diluted with PBS, and 10 ml of 22.5% stock Percoll diluted with PBS. Five milliliters of the spore suspension was layered over the gradient into a 50-ml Falcon centrifuge tube. After centrifugation at 2,500⫻g
for 30 min at 15°C, four distinct bands were formed. The clearly defined ring at the 90 to 67.5% Percoll interface, previously determined to contain whole spores by light microscopy and TEM (results not shown), was collected, washed three times in PBS, pelleted at 2,500⫻gfor 20 min, and resuspended in PBS (1/3 [vol/vol]).
(iii) Sterilization.To monitor for bacterial and fungal contaminants, the iso-late of spores was mixed with an antibiotic solution of ceftriaxone (20g/ml), vancomycin (10g/ml), amikacin (10g/ml), and amphotericin B (0.25g/ml) and placed at 4°C. Antibiotics were added daily at the same concentration until sterilization of the preparation as determined by aerobic and anaerobic cultiva-tion was achieved. After 3 to 5 days of this regimen, the sterile concentrate was centrifuged at 2,500⫻gfor 20 min and washed twice in sterile PBS.
The pellet was then divided into two aliquots of 1 ml each in sterile PBS, in one of which spores were incubated for 60 min at room temperature with 50l of a concentrated (5 IU/ml) chitinase fromSerratia marcescens(Sigma Laboratories), treated by two freeze-thaw cycles, centrifuged at 2,500 ⫻g for 20 min, and examined after fluorochrome Uvitex 2B stain.
Aliquots were resuspended and diluted in sterile 0.15 M NaCl (1/3 [vol/vol]). Spore counts were performed by using 2-l droplets applied to 5-mm-wide wells on multiwell slides, stained according to the Uvitex 2B method.
Production of MAbs.Adult (6-week-old) female BALB/c mice, for hybridoma development and ascites production, were purchased from Charles River Lab-oratories (Saint-Aubin-les-Elbeuf, France). Two protocols of immunization, us-ing two groups of six mice each, were carried out. In protocol 1, animals received whole spores ofE. bieneusi; in protocol 2, they received chitinase-treated spores. All the animals were immunized intraperitoneally (i.p.) four times at 3-week intervals with 5.6⫻107spores per 100l emulsified at a 1:1 ratio in Freund’s
complete adjuvant (Sigma Laboratories) for the first inoculation and in Freund’s incomplete adjuvant (Sigma Laboratories) for the other three inoculations. Two control mice were not injected.
Seven days after each immunization, sera were screened by indirect immuno-fluorescence as described below to determine which mice had the highest para-site-specific antibody responses. Mouse serum adsorption experiments were per-formed with an antigen preparation of enteropathogenic bacteria and yeasts isolated from human stool samples and grown on aerobic culture. The prepara-tion was added to serum samples prediluted in PBS, which subsequently were shaken at room temperature for 120 min and spun down (10,000⫻g, 5 min). Measurements were done by using the supernatants. Sera were stored at⫺80°C and used as positive controls during all immunoassays.
Two of the immunized mice, one in each group, were selected to receive a further intravenous dose of 2⫻107spores in 100l of sterile 0.15 M NaCl, and
their spleens were used for the fusion protocol 3 days later (one fusion protocol per immunization protocol). Cells of the murine myeloma line SP2/O were fused with spleen cells from the donor mouse at a 1:5 ratio in 50% polyethylene glycol (Sigma Laboratories) (13). Stable hybrids were selected by growth in Dulbecco’s minimum essential medium containing 20% fetal bovine serum, hypoxanthine, aminopterin, and thymidine as previously described (33). Supernatant culture medium was screened by an indirect immunofluorescence antibody test (IFAT). Hybridoma cultures whose supernatants showed antibody activity againstE. bieneusiwere expanded onto 24-well plates and cloned twice by limiting dilutions (13). Pristane-primed female BALB/c mice were injected i.p. with 2⫻106cells
from each hybridoma line, and ascitic fluid was collected 10 to 15 days later, centrifuged (400⫻gfor 15 min) to remove cells, aliquoted, and stored at⫺80°C until used (13). Culture supernatants of the different hybridoma lines were also collected. MAbs were purified from ascites or supernatants with Dynabeads M-450 rat anti-mouse immunoglobulin M (IgM) and M-450 rat anti-mouse IgG2a (Dynal, Compie`gne, France), according to the manufacturer’s instruc-tions.
IFAT.The IFAT was performed with (i) washed whole spores ofE. bieneusi, which were used for the immunization in protocol 1, suspended in PBS to obtain 108spores per ml and (ii)E. intestinalisspores from tissue culture supernatants
resuspended in PBS at 109spores per ml, as antigens. Antigen slides were
prepared by depositing 2-l volumes of the spore suspension onto each well of 18-well slides, which were then air dried and fixed in ice-cold acetone for 10 min. Undiluted supernatants of hybridoma cultures or the ascitic fluid, serially diluted twofold in 0.1% bovine serum albumin (BSA) in PBS starting from a 1:2 dilution, were transferred to the 18-well slides (20l of each dilution per well),
and the slides were incubated at room temperature for 30 min in a moist chamber. The slides were then washed three times in PBS at 10-min intervals, and each well was then covered with 20l of fluorescein isothiocyanate (FITC)-labelled goat anti-mouse IgG-IgM-IgA (Sigma Laboratories) at a dilution of 1:200 containing Evans blue as the counterstain. The slides were incubated at room temperature for 30 min and washed three times as described above, coverslips were added with buffered glycerol mounting fluid, and the slides were examined with a Leitz Laborlux fluorescence microscope equipped with epiflu-orescence illumination. PBS and conjugate controls were included with each slide.
Characterization of anti-E. bieneusiMAbs. (i) Isotyping.The MAb isotypes were determined with a dipstick isotyping kit (Sigma Laboratories) according to the instructions enclosed.
(ii) Ultrastructural immunolocalization.Stool samples from patients with an
E. bieneusiinfection were purified by gentle filtration and Percoll discontinuous gradient as described earlier. Then they were fixed at room temperature in 4% paraformaldehyde–0.5% glutaraldehyde in 0.15 M Na cacodylate buffer (pH 7.2) for 45 min and rinsed three times at 10-min intervals in 0.1 M ammonium chloride in cacodylate buffer and one time for 10 min in cacodylate buffer alone. After ethanolic dehydration, the material was embedded in LR WHITE resin. The sections were collected on gold or nickel grids. They were incubated at room temperature in 1% BSA in PBS for 45 min to block unbound sites and then for 120 min with ascitic fluid containing MAbs (1:512). After a series of six washings (5 min each), in 0.25% BSA in PBS, sections were incubated for 60 to 120 min with goat anti-mouse affinity-purified IgM or goat anti-mouse affinity-purified IgG labelled with 10-m gold particles (Sigma Laboratories) used as second antibody. Controls consisted of sections incubated with the second antibody alone. After being washed in sterilized water, samples were examined with a JEOL JEM 100 CX transmission electron microscope.
(iii) SDS-PAGE and Western blot analysis.Western blot analysis was per-formed withE. intestinalisspores used as antigens. Parasite proteins were sepa-rated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (18), with a 5% stacking gel and a 12% resolving gel. Intact spores in 1 ml of sample buffer containing 5% -mer-captoethanol were boiled for 5 min and centrifuged at 10,000⫻gto remove particulate materials. Each preparative slab gel (16 by 20 cm) was loaded with 2⫻109parasites. After electrophoresis, the separated polypeptides were
elec-trophoretically transferred onto a nitrocellulose membrane (pore diameter, 0.22
m; Bio-Rad, Ivry-sur-Seine, France) which was then incubated with 5% (wt/vol) nonfat dry milk (Re´gilait) in PBS for 60 min to block unbound sites, washed in PBS containing 0.05% Tween 20 (PBS-Tween) for 20 min and cut into 3-mm-wide longitudinal strips. The strips were incubated for 60 min with either undi-luted supernatants or ascitic fluid containing MAbs (1:512), murine immune sera, preimmune murine sera, or a specificE. intestinalisIgG3 6C12C11 MAb previously developed in our laboratory (unpublished data) as a positive control. After being washed in PBS-Tween, strips were incubated for 60 min with affinity-purified peroxidase-labelled goat anti-mouse IgG-IgM antiserum (Sigma ratories) diluted 1:2,000 and developed with 4-chloro-1-naphthol (Sigma Labo-ratories) after being washed with three changes of PBS-Tween. After color development for 30 min, the strips were rinsed in distilled water, dried, and stored in the dark.
(iv) Cross-reactivity studies.The reactivity of the MAbs with bacteria, para-sites, and fungi was assessed by IFAT.
Utilization of MAbs in diagnosis and comparison with other methods.Fifteen diarrheal fecal samples containing microsporidial spores and 25 negative stool samples were collected and preserved in PBS with 10% Formol (1/3 [vol/vol]). Intestinal microsporidiosis had been previously diagnosed in the 15 patients by classical staining methods (16, 31). The fecal samples were filtered through a 50-m-pore-diameter filter, and after ether sedimentation by centrifugation at 2,500⫻gfor 15 min, the pellet was suspended in PBS (1/3 [vol/vol]) and applied to slides. All the stool samples were coded and blind-tested by IFAT. The specificities of the MAbs were evaluated by comparison with PCR and TEM (if available) performed as described previously.
RESULTS
MAb production.
Prior to immunization, mouse sera were
screened for antibodies against intestinal microsporidium
spores by IFAT and Western blot analysis, with
E. bieneusi
and
E. intestinalis
spores as antigens. Preimmunization sera and
healthy-mouse-control sera did not react with any of the
par-asite spores. Because they were immunized with impure
anti-gens, BALB/c mice were screened for serum parasite-specific
antibody response 7 days after each injection before a final
boost and fusion. All mice began to produce antibodies after
the second i.p. injection. The highest antibody response was
raised after the fourth injection. Sera from mice 2-1 (protocol
1) and 3-2 (protocol 2) produced the better fluorescence
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iting dilution, 1:100). These two mice were thus selected for the
fusion protocol.
For each immunization protocol, a single fusion was
per-formed with spleen cells of the donor mouse. A total of 960
wells were seeded with fused SP2/O myeloma cells, and their
supernatants began to be screened by whole spore
E. bieneusi
IFAT 11 days after fusion. After the third screening, six
anti-body-secreting hybridomas still reacted against spores of
E.
bieneusi
. After two cloning procedures by limiting dilutions,
two stable clones, clone 3B82H2 from protocol 1 and clone
6E52D9 from protocol 2, were obtained. Isotype
determina-tion revealed that clone 3B82H2 secreted IgM and clone
6E52D9 secreted IgG2a. The two MAbs were expanded in
both ascites and culture and subjected to thorough screening.
IFAT.
The two MAbs showed strong indirect
immunofluo-rescence after incubation with whole purified
E. bieneusi
spores, almost to the same extent, and yielded titers of greater
than 4,096. All reacted exclusively with the walls of the spores,
which fluoresced brightly and were thus easily recognized with
a
⫻
1,000 magnification (Fig. 1A and B). 3B82H2 and 6E52D9
showed mutual competition in their binding to
E. bieneusi
when the two MAbs were combined in one IFAT. No
fluores-cence was observed on
E. intestinalis
spore walls or filaments or
when FITC-conjugated second antibody was employed alone.
Characterization of the MAbs. (i) Ultrastructural
immuno-localization.
The binding of each MAb to
E. bieneusi
spores
was studied by TEM which revealed a labelling of the spore
wall, more especially when sections of mature spores were
incubated with 3B82H2 or 6E52D9 MAb. Interestingly,
differ-ent parts of the spore wall reacted with these MAbs. Gold
particles were exclusively distributed over the exospore in
spec-imens treated with the IgG MAb 6E52D9 (Fig. 2A and B),
whereas a labelling of the endospore was obtained with the
IgM MAb 3B82H2 (Fig. 2C and D). No labelling was observed
when sections were incubated with the second antibody alone.
No reactivity was displayed by
E. intestinalis
spores collected in
culture supernatants or in stool samples. The immunogold
labelling of the spore wall was consistent with the IFAT results.
(ii) Western blot analysis.
Neither of the two MAbs reacted
with
E. intestinalis
antigens by Western blot analysis, compared
to an IgG3 6C12C11 MAb directed against
E. intestinalis
whole
spores.
(iii) Cross-reactivity studies.
By IFAT, MAbs in ascitic fluid
were assessed for cross-reactivity to enteropathogenic
bac-teria (
Escherichia coli
,
Proteus vulgaris
,
Klebsiella pneumoniae
,
Shigella dysenteriae
,
Salmonella typhi
,
Yersinia enterocolitica
,
Pseudomonas aeruginosa
,
Enterobacter aerogenes
,
Enterococcus
faecalis
), other intestinal parasites (
Giardia intestinalis
,
Entam-oeba histolytica
,
Entamoeba coli
,
Cryptosporidium parvum
,
Sar-cocystis hominis
,
Isospora belli
,
Blastocystis hominis
), and yeasts
from stool samples. There was no cross-reactivity even at the
1:128 dilution to any of the organisms evaluated.
Utilization of MAbs in diagnosis and comparison with other
methods.
By Uvitex 2B and Weber’s modified trichrome
stain-ing methods, spores with the morphological features
charac-teristic of microsporidia (16, 31) were detected in fecal
speci-mens from 15 patients. Among these samples, 14 contained
small oval spores suggestive of
E. bieneusi
, whereas 1 stool
contained larger spores suggestive of
E. intestinalis
.
By IFAT, the two MAbs reacted exclusively with the small
oval spores present in the 14 samples (Table 1). These spores
fluoresced brightly, 4
⫹
in a scale of 0 to 4, with a prominent
labelling of the spore walls when smears were treated with
3B82H2 or 6E52D9 MAb at 1:512 and 1:1,024 dilutions (Fig.
1C). Neither of the two MAbs generated background in
for-malin-fixed stool specimens. No fluorescence was observed
either in the stool containing larger spores suggestive of
E.
intestinalis
or in stools from patients without intestinal
micro-sporidiosis.
The IFAT was compared for reliability with the PCR and
TEM (if available). The results were consistent with those of
IFAT (Table 1). The microsporidian species of each positive
sample was confirmed and
E. bieneusi
but not
E. intestinalis
[image:3.612.322.539.71.554.2]spores were recognized by MAbs 6E52D9 and 3B82H2. By
PCR, no signals were observed for the 25 fecal specimens
negative for microsporidia.
FIG. 1. Purified whole spores ofE. bieneusistained by indirect immunoflu-orescence with MAbs 6E52D9 (A) and 3B82H2 (B) in ascitic fluid. MAbs recognize antigens localized in the spores walls. (C) Formalin-fixed smear of a fecal sample, from one of the 14 patients with microsporidia, reacted with a 1:512 dilution of MAb 6E52D9. Note the bright fluorescence of spore walls. Bar⫽5
m.
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DISCUSSION
[image:4.612.74.293.72.574.2]We report here the production, characterization, and
reac-tivity of the first MAbs directed against
E. bieneusi
, the most
common microsporidium infecting AIDS patients. Since there
FIG. 2. Immunogold electron micrographs ofE. bieneusimature spores after incubation with MAb 6E52D9 and MAb 3B82H2. (A) Labelling of the outer layer of the spore wall (exospore [arrowhead]) with MAb 6E52D9. The arrow indicates the double row arrangement ofE. bieneusipolar tube sections. Mag-nification,⫻120,000. (B) The MAb selectively labels the exospore (arrowhead). No gold particles are visible at the surface or inside the bacteria (left). Magni-fication,⫻100,000. (C) The tangential section of the spore treated with MAb 3B82H2 shows the distribution of gold particles in the internal layer of the wall (endospore [arrowhead]). Magnification,⫻100,000. (D) Labelling of the endo-spore (arrowhead) with the same MAb. Magnification,⫻100,000.
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is no in vitro culture system presently available, it has been
impossible to produce enough antigen to screen for specific
antibodies. We circumvented this problem by developing a
procedure for the isolation, purification, and sterilization of
parasite spores from human stools. Apparently, the best
pres-ervation of the spore antigens is obtained when using gentle
filtration, centrifugation in isotonic conditions, and gradual
addition of low concentrations of antibiotics to the final fecal
suspension.
The most difficult part of the study was identification of an
MAb with the desired specificity because immunization was
done with impure antigenic material and assays to detect the
desired MAb were not available. However, the IFAT
per-formed with
E. bieneusi
-purified whole spores as antigens
ap-peared to be specific enough to select the MAbs that bound to
spore walls, and the results of the immunofluorescence assays
could be confirmed at the ultrastructural level by TEM.
Of the two MAbs, one belongs to the IgM class (protocol 1)
and the other belongs to the IgG class (protocol 2). The
spe-cific reactivity of the selected antibodies depends on the nature
of the immunogen. Significantly, the MAb 3B82H2 reactive
with the endospore known to contain chitin was raised against
the antigenic fraction which was not treated with chitinase.
Extensive cross-reactivities among different microsporidian
species have been observed with polyclonal sera from rabbits
immunized with a single microsporidian species (22). More
recently, polyclonal antisera raised against
Encephalitozoon
cu-niculi
in rabbits or in mice were used in the IFAT to detect
E.
bieneusi
organisms in deparaffinized tissue sections (36) and in
stool (3, 39). Using the MAbs described in this study in either
IFAT, TEM or Western blot analysis, we did not observe any
cross-reactions with the other human intestinal
microspo-ridium,
E. intestinalis
, or with other intestinal parasites, yeast
cells, or bacteria.
The reference techniques used for the comparative
detec-tion of microsporidia directly from fecal specimens were PCR,
TEM, and Uvitex 2B and Weber’s modified trichrome
stain-ings. Immunofluorescence assay with MAbs appeared to be
highly specific.
E. bieneusi
spores were identified in the 14 stool
specimens with complete concordance with the results of PCR
and TEM (if available). The sensitivities of the MAbs in
de-tecting subclinical infections seemed attractive. Indeed, the
diagnosis could be performed even when few spores were
ex-creted in stool samples. It is noteworthy that no background
was observed in the fecal specimens which were examined by
the immunofluorescence protocol described herein. Moreover,
the application of these MAbs as tools for detecting
E. bieneusi
in feces does not require either pretreatment of the samples or
absorbing the FITC-conjugated antibodies with formalin-fixed
stool sediment, as described by others (3, 5).
Diagnosis of microsporidiosis to the species level is essential
for the treatment of patients. To date, although different
ther-apeutic agents are effective against most microsporidian
spe-cies, none can eradicate
E. bieneusi
except fumagillin, which is
still under investigation (20). Presently, the identification of
human microsporidia to the species level requires
time-con-suming methods such as electron microscopy and molecular
techniques (6, 7, 9, 11, 23, 35). The two MAbs newly described
in this study are the first to be raised against
E. bieneusi
. Their
application in the immunofluorescence identification of this
organism could offer a new diagnostic tool for clinical
labora-tories. The bright fluorescence of the spore wall facilitates the
diagnosis even for the untrained eye. In addition these MAbs
could offer new approaches for the study of
E. bieneusi
. Used
as ligands, they could enable the isolation and purification of
E.
bieneusi
spores with methods such as affinity chromatography
or immunomagnetic separation. Ongoing studies will
deter-mine the usefulness of these techniques in our follow-up assays
to develop in vivo (1, 2) or in vitro models of
E. bieneusi
as well
as in Western blot analysis or enzyme-linked immunosorbent
assay.
ACKNOWLEDGMENTS
We thank Jean Jacques Hauw for providing TEM facilities and
Jacques Breton for revising the manuscript.
[image:5.612.53.552.84.269.2]This study was supported by grants from SIDACTION.
TABLE 1. Comparison of four diagnostic methods for the 15 patients with microsporidia detected by stained smears of stool samples
aCase
no. Sexc Age(yr) statusHIV CD4/mmNo. of3
Stool sample results by: Light microscopyb PCRd
TEM IFAT Small Large E. bieneusi E. intestinalis 3B82H2 6E52D9
1
M
30
⫹
44
N
⫹
⫺
ND
⫹
⫹
2
M
16
⫺
737
VN
⫹
⫺
E. bieneusi
⫹
⫹
3
M
52
⫹
0
R
⫹
⫺
E. bieneusi
⫹
⫹
4
M
42
⫹
119
F
⫹
⫺
E. bieneusi
⫹
⫹
5
F
26
⫹
13
N
⫹
⫺
E. bieneusi
⫹
⫹
6
M
38
⫹
35
N
⫹
⫺
ND
⫹
⫹
7
M
39
⫹
23
R
⫹
⫺
ND
⫹
⫹
8
M
29
⫹
10
N
⫹
⫺
E. bieneusi
⫹
⫹
9
F
35
⫹
3
VN
⫹
⫺
E. bieneusi
⫹
⫹
10
M
39
⫹
41
R
⫺
⫹
E. intestinalis
⫺
⫺
11
M
49
⫹
20
N
⫹
⫺
ND
⫹
⫹
12
M
50
⫺
ND
N
⫹
⫺
ND
⫹
⫹
13
M
52
⫹
6
N
⫹
⫺
E. bieneusi
⫹
⫹
14
M
45
⫹
40
F
⫹
⫺
ND
⫹
⫹
15
M
34
⫹
15
VN
⫹
⫺
ND
⫹
⫹
a⫹, positive;⫺, negative; ND, not done.
bUvitex 2B stain and Weber’s modified trichrome stain were performed for all patients. Spores were classified as either small (diameter, 1 to 1.5m) or large
(diameter, 1.2 to 2.2m). Classification of spore quantity per microscopic field (magnification,⫻1,000; oil immersion): VN, very numerous; N, numerous; F, few; R, rare.
cM, male; F, female.
dSpecific PCR assay for direct detection of intestinal microsporidia.
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