Detection of Microsporidia by Indirect Immunofluorescence
5
8/20/2019 Detection of Microsporidia by Indirect Immunofluorescence http://slidepdf.com/reader/full/detection-of-microsporidia-by-indirect-immunofluorescence 1/5 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1994, p. 608-612 0095-1137/94/ 04.00+0 Copyright © 1994, American Society for Microbiology Detection of Microsporidia by Indirect Immunofluorescence Antibody Test Using Polyclonal and Monoclonal Antibodies ABDALLA M. ALDRAS,' JAN M. ORENSTEIN,2 DONALD P. KOTLER,3 JOHN A. SHADDUCK,4 AND ELIZABETH S. DIDIER1* Department of Microbiology, Tulane University Regional Primate Research Center, Covington, Louisiana 704331; Department of Pathology, George Washington University Medical Center, Washington, D.C. 200372; St. Luke's/Roosevelt University Medical Center, Ne w York, New York 100253; and Department of Veterinary Pathobiology, Texas A MUniversity, College Station, Texas 778434 Received 5 August 1993/Returned for modification 4 October 1993/Accepted 17 November 1993 During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and Ell, and were found to secrete MAbs of the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. helkm antigens with relative molecular weights (Mrs) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with Mrs of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. helem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. heUlem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. helkm, ca n be used for indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients. Microsporidia are small obligate intracellular protozoan parasites which infect a wide range of animal hosts, including all classes of vertebrates and most invertebrates (8). Awareness of microsporidia is important because the number of reported microsporidiosis cases in AIDS patients is increasing (2, 5-7, 22, 23, 29). The three most common microsporidia reported to infect individuals with AIDS are Enterocytozoon bieneusi, En- cephalitozoon hellem, and the Encephalitozoon-like Septata intestinalis. E. bieneusi primarily infectssmall-intestinal entero- cytes, causing diarrhea, but can also infect the biliary tract, leading to cholangitis (4, 10, 19, 23, 26). On the other hand, E. hellem and the Encephalitozoon-like S. intestinalis are not as tissue specific and have been reported to cause keratoconjunc- tivitis, sinusitis, nephritis, and enteritis (3, 15, 22, 27). Although cases of Encephalitozoon cuniculi-associated hepatitis and peri- tonitis in AIDS patients have been reported, it is possible that they were actually due to the morphologically identical E. hellem. Presently, definitive identification of microsporidiosis de- pends upon transmission electron microscopy, which is time- and cost-consuming. In addition, transmission electron micros- copy may not be sensitive enough to detect small numbers of organisms. Serological studies for detecting microsporidium- specific antibodies are reliable for antemortem diagnosis in infected laboratory animals (29, 30) but may be unreliable for Corresponding author. AIDS patients whose immune responses are compromised (12, 13). Mammalian microsporidium spores do stain with Gram, Giemsa, calcofluor, and concentrated trichrome (25, 30, 32, 33, 35), butbecause these organisms are very small (measuring 0.5 to 2.0 ,um by 1.0 to 4.0 ,um) they are difficult to distinguish from bacteria and small yeasts. In this article, we report the use of polyclonal antibodies and monoclonal antibodies (MAbs) in detecting microsporidia in formalin-fixed stool and urine by the indirect immunofluorescence antibody test (IFAT). MATERIALS N METHODS Parasites. E. hellem, E. cuniculi, and Nosema corneum were grown in Madin-Darby canine kidney (MDCK) cells by using RPMI 1640 supplemented with 5 fetal bovine serum, strep tomycin (100 ,ug/ml), penicillin (100 U/ml), amphotericin B (0.25 ,ug/ml), and L-glutamine (2 mM) (referred to as complete RPMI) as described previously (11, 14). Parasites from tissue culture supernatants were centrifuged at 400 x g for 15 min, washed once in 25 mM Tris-buffered saline (TBS; pH 7.4) containing 0.05 Tween 20 (TBS-Tween), resuspended in TBS, and centrifuged over 50 Percoll (Pharmacia, Piscat- away, N.J.) at 14,000 x g for 2 min. The parasites were washed twice with TBS to remove the Percoll and stored at 4°C in bicarbonate buffer (pH 9.6) for us e in the enzyme-linked immunosorbent assay (ELISA) or were stored in TBS for all other uses. Antibodies. Murine hyperimmune antisera were obtained 60 8 Vol. 32, No. 3
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Transcript of Detection of Microsporidia by Indirect Immunofluorescence
8/20/2019 Detection of Microsporidia by Indirect Immunofluorescence
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J O U R N A L
OF C L I N I C A L
M I C R O B I O L O G Y ,
M a r .
1 9 9 4 , p .
6 0 8 - 6 1 2
0 0 9 5 - 1 1 3 7 / 9 4 / 0 4 . 0 0 + 0
C o p y r i g h t
©
1 9 9 4 , A m e r i c a n
S o c i e t y f o r
M i c r o b i o l o g y
D e t e c t i o n
o f M i c r o s p o r i d i a
b y
I n d i r e c t
I m m u n o f l u o r e s c e n c e
A n t i b o d y
T e s t
U s i n g
P o l y c l o n a l a n d
M o n o c l o n a l
A n t i b o d i e s
ABDALLA M .
ALDRAS,' JAN M.
ORENSTEIN,2
DONALD P . KOTLER,3 JOHN
A . SHADDUCK,4
AND
ELIZABETH
S .
DIDIER1*
D e p a r t m e n t
o f M i c r o b i o l o g y , T u l a n e
U n i v e r s i t y R e g i o n a l
P r i m a t e R e s e a r c h
C e n t e r , C o v i n g t o n ,
L o u i s i a n a
7 0 4 3 3 1 ;
D e p a r t m e n t
o f P a t h o l o g y ,
G e o r g e
W a s h i n g t o n U n i v e r s i t y M e d i c a l
C e n t e r ,
W a sh i n gt o n , D . C .
2 0 0 3 7 2 ;
S t . L u k e ' s / R o o s e v e l t
U n i v e r s i t y
M e d i c a l C e n t e r ,
Ne w
Y o r k ,
New
Y o r k
1 0 0 2 5 3 ;
a n d
D e p a r t m e n t
o f
V e t e r i n a r y P a t h o b i o l o g y ,
T e x a s
A
M
U n i v e r s i t y ,
C o l l e g e
S t a t i o n , T e x a s 7 7 8 4 3 4
R e c e i v e d
5
A u g u s t
1 9 9 3 / R e t u r n e d
f o r m o d i f i c a t i o n
4
O c t o b e r
1 9 9 3 / A c c e p t e d 1 7 N o v e m b e r 1 9 9 3
D u r i n g a
s c r e e n i n g f o r
m o n o c l o n a l
a n t i b o d i e s ( M A b s ) t o
t h e
m i c r o s p o r i d i a n
E n c e p h a l i t o z o o n h e l l e m , t h r e e
m u r i n e
h y b r i d o m a
c e l l l i n e s
p r o d u c i n g s t r o n g
e n z y m e - l i n k e d
i m m u n o s o r b e n t
assay
( E L I S A )
r e a c t i v i t i e s
were
c l o n e d
t w i c e ,
were d e s i g n a t e d
C 1 2 , E 9 , a n d E l l , a n d
were f o u n d t o s e c r e t e MAbs
o f t h e i m m u n o g l o b u l i n
M
i s o t y p e . On
s u b s e q u e n t E L I S A s , t h e t h r e e
M A b s r e a c t e d
most s t r o n g l y t o E .
h e l l e m , a n d t h e y
r e a c t e d s o m e w h a t
l e s s t o
E n c e p h a l i t o z o o n
c u n i c u l i a n d
l e a s t t o N o s e m a
corneum, t w o
o t h e r m i c r o s p o r i d i a n
s p e c i e s . T h e
MAbs
p r o d u c e d
v a l u e s o f
a b s o r b a n c e a g a i n s t
m i c r o s p o r i d i a t h a t were
a t l e a s t t h r e e
t i m e s g r e a t e r t h a n
r e a c t i v i t i e s
o b t ai n e d w i t h c o n t r o l
h y b r i d o m a
s u p e r n a t a n t s o r
w i t h
u n i n f e c t e d
h o s t c e l l
p r o t e i n s
u s e d
as
a n t i g e n s .
B y
W e s t e r n
b l o t
i m m u n o d e t e c t i o n ,
t h e t h r e e
MAbs
d e t e c t e d t h r e e
E .
h e l k m
a n t i g e n s
w i t h
r e l a t i v e
m o l e c u l a r
w e i g h t s
( M r s )
o f
6 2 ,
6 0 , a n d
5 2 w h e n
a s s a y e d
a t t h e
h i g h e s t s u p e r n a t a n t
d i l u t i o n s
p r o d u c i n g
r e a c t i v i t y .
A t
l o w e r
d i l u t i o n s , t h e
M A bs
d e t e c t e d
a d d i t i o n a l
p r o t e i n s w i t h
M r s
o f
5 5 a n d 5 3 .
B y u s i n g i n d i r e c t i m m u n o f l u o r e s c e n c e
a n t i b o d y
s t a i n i n g ,
t h e
M A b s ,
as
w e l l as
h y p e r i m m u n e
p o l y c l o n a l
m u r i n e
a n t i s e r a r a i s e d
a g a i n s t
E .
c u n i c u l i a n d
E .
h e l e m ,
were a b l e t o d e t e c t
f o r m a l i n - f i x e d ,
t i s s u e c u l tu r e- d er i v e d E .
c u n i c u l i a n d E .
h e U l e m a n d
t w o o t h er
human
m i c r o s p o r i d i a , E n t e r o c y t o z o o n
b i e n e u s i a n d S e p t a t a
i n t e s t i n a l i s ,
i n f o r m a l i n - f i x e d
s t o o l a n d u r i n e ,
r e s p e c t i v e l y . E .
b i e n e u s i ,
h o w e v e r , s t a i n e d
more
i n t e n s e l y
w i t h
t h e p o l y cl o n al a n ti s e r a
t h a n w i t h t h e
M A b s .
N e i t h e r
t h e MAbs no r t h e
h y p e r i m m u n e m u r i n e p o l y c l o n a l
a n t i b o d i e s d e t e c t e d
C r y p t o s p o r i d i u m , G i a r d i a ,
T r i c h o m o n a s , or
I s o s p o r a
spp.
A t h i g h e r
c o n c e n t r a t i o n s ,
t h e
p o l y c l o n a l
a n t i s e r a d i d s t a i n
N . corneum a n d y e a s t
c e l l s .
T h e
b a c k g r o u n d s t a i n i n g
c o ul d b e
a b s o r b e d
w i t h
C a n d i d a
a l b i c a n s .
T h e s e
r e s u l t s
d e m o n s t r a t e t h a t
p o l y c l o n a l a n t i s e r a t o E .
c u n i c u l i a n d E .
h e l l e m ,
as w e l l as MAbs
r ai s ed a g a in s t E .
h e l k m ,
ca n
b e u s e d
f o r
i n d i r e c t
i m m u n o f l u o r e s c e n c e
a n t i b o d y s t a i n i n g t o d e t e c t s e v e r a l
s p e c i e s o f
m i c r o s p o r i d i a
k n o w n
t o cause
o p p o r t u n i s t i c
i n f e c t i o n s i n AIDS p a t i e n t s .
M i c r o s p o r i d i a are s m a l l
o b l i g a t e
i n t r a c e l l u l a r p r o t o z o a n
p a r a s i t e s
w h i c h
i n f e c t
a w i d e
range
o f
a n i m a l
h o s t s ,
i n c l u d i n g
a l l
c l a s s e s
o f
v e r t e b r a t e s a n d most
i n v e r t e b r a t e s ( 8 ) .
A w a r e n e s s
o f
m i c r o s p o r i d i a
i s
i m p o r t a n t
b e c a u s e
t h e
number
o f
r e p o r t e d
m i c r o s p o r i d i o s i s
cases
i n
AIDS
p a t i e n t s i s i n c r e a s i n g ( 2 , 5 - 7 ,
2 2 , 2 3 , 2 9 ) . T h e t h r e e
most common
m i c r o s p o r i d i a
r e p o r t e d t o
i n f e c t
i n d i v i d u a l s w i t h
AIDS
are E n t e r o c y t o z o o n
b i e n e u s i ,
E n -
c e p h a l i t o z o o n
h e l l e m , a n d
t h e
E n c e p h a l i t o z o o n - l i k e S e p t a t a
i n t e s t i n a l i s .
E . b i e n e u s i
p r i m a r i l y i n f e c t s s m a l l - i n t e s t i n a l
e n t e r o -
c y t e s ,
c a u s i n g d i a r r h e a , b u t can a l s o i n f e c t t h e
b i l i a r y t r a c t ,
l e a d i n g
t o
c h o l a n g i t i s
( 4 , 1 0 , 1 9 , 2 3 ,
2 6 ) . On
t h e o t h e r
h a n d , E .
h e l l e m
a n d
t h e E n c e p h a l i t o z o o n - l i k e
S .
i n t e s t i n a l i s are n o t as
t i s s u e
s p e c i f i c
a n d
h a v e
b e e n
r e p o r t e d
t o
cause
k e r a t o c o n j u n c -
t i v i t i s , s i n u s i t i s ,
n e p h r i t i s ,
a n d e n t e r i t i s
( 3 , 1 5 , 2 2 ,
2 7 ) .
A l t h o u g h
cases
o f
E n c e p h a l i t o z o o n
c u n i c u l i - a s s o c i a t e d
h e p a t i t i s
a n d
p e r i -
t o n i t i s i n
AIDS
p a t i e n t s
h a v e b e e n
r e p o r t e d ,
i t i s
p o s s i b l e
t h a t
t h e y
were
a c t u a l l y
d u e
t o
t h e
m o r p h o l o g i c a l l y
i d e n t i c a l
E .
h e l l e m .
P r e s e n t l y ,
d e f i n i t i v e
i d e n t i f i c a t i o n o f
m i c r o s p o r i d i o s i s
d e -
p e n d s
upon
t r a n s m i s s i o n
e l e c t r o n
m i c r o s c o p y ,
w h i c h
i s
t i m e -
a n d
c o s t - c o n s u m i n g .
I n
a d d i t i o n ,
t r a n s m i s s i o n e l e c t r o n
m i c r o s -
copy
may
n o t
b e
s e n s i t i v e
e n o u g h
t o
d e t e c t s m a l l n u m b e r s o f
o r g a n i s m s . S e r o l o g i c a l s t u d i e s f o r
d e t e c t i n g
m i c r o s p o r i d i u m -
s p e c i f i c
a n t i b o d i e s
are r e l i a b l e
f o r antemortem
d i a g n o s i s
i n
i n f e c t e d
l a b o r a t o r y
a n i m a l s
( 2 9 ,
3 0 ) b u t may b e u n r e l i a b l e
f o r
C o r r e s p o n d i n g
a u t h o r .
AIDS
p a t i e n t s w h o s e immune
responses
are
c o m p r o m i s e d
( 1 2 ,
1 3 ) .
Mammalian
m i c r o s p o r i d i u m spores
d o
s t a i n
w i t h
G r a m ,
G i e m s a , c a l c o f l u o r ,
a n d c o n c e n t r a t e d
t r i c h r o m e
( 2 5 , 3 0 , 3 2 , 3 3 ,
3 5 ) ,
b u t b e c a u s e
t h e s e
o r g a n i s m s
are
very
s m a l l
( m e a s u r i n g
0 . 5
t o 2 . 0
, u m
b y
1 . 0 t o 4 . 0
, u m )
t h e y
are d i f f i c u l t
t o
d i s t i n g u i s h f r o m
b a c t e r i a a n d
s m a l l
y e a s t s .
I n
t h i s
a r t i c l e ,
we
r e p o r t
t h e
use
o f
p o l y c l o na l a nt i b o d i e s
a n d m o n o c l o n a l
a n t i b o d i e s
( M A b s ) i n
d e t e c t i n g
m i c r o s p o r i d i a
i n
f o r m a l i n - f i x e d s t o o l a n d
u r i n e
b y
t h e
i n d i r e c t i m m u n o f l u o r e s c e n c e
a n t i b o d y
t e s t
( I F A T ) .
M A T E RI ALS N
METHODS
P a r a s i t e s .
E .
h e l l e m ,
E .
c u n i c u l i ,
a n d Nosema corneum
were
grown
i n
M a d i n - D a r b y
c a n i n e
k i d n e y (MDCK)
c e l l s
b y u s i n g
RPM I
1 6 4 0
s u p p l e m e n t e d
w i t h 5
f e t a l b o v i n e
serum,
s t r e p
t o m y c i n ( 1 0 0
, u g / m l ) ,
p e n i c i l l i n
( 1 0 0
U / m l ) , a m p h o t e r i c i n
B
( 0 . 2 5 , u g / m l ) ,
a n d
L - g l u t a m i n e
( 2 m M )
( r e f e r r e d
t o as
c o m p l e t e
RPMI)
as d e s c r i b e d
p r e v i o u s l y
( 1 1 , 1 4 ) .
P a r a s i t e s f r o m
t i s s u e
c u l t u r e
s u p e r n a t a n t s
were
c e n t r i f u g e d
a t 4 0 0
x
g
f o r
1 5
m i n ,
w a s h e d
once
i n
2 5 mM
T r i s - b u f f e r e d
s a l i n e
( T B S ; pH
7 . 4 )
c o n t a i n i n g
0 . 0 5
Tween 2 0
( T B S - T w e e n ) ,
r e s u s p e n d e d
i n
T B S ,
a n d
c e n t r i f u g e d
ov er 50 P e r c o l l
( P h a r m a c i a ,
P i s c a t -
away, N . J . )
a t
1 4 , 0 0 0
x
g
f o r 2
m i n .
The
p a r a s i t e s
were
w a s h e d
t w i c e
w i t h
TBS t o
remove t h e P e r c o l l
and
s t o r e d
a t
4 ° C
i n
b i c a r b o n a t e b u f f e r
( p H 9 . 6 )
f o r use i n
t h e
e n z y m e - l i n k e d
i m m u n o s o r b e n t
assay
( E L I S A )
or were s t o r e d
i n
TBS f o r
a l l
o t h e r uses.
A n t i b o d i e s .
M u r i n e
h y p e r i m m u n e
a n t i s e r a
were
o b t a i n e d
60 8
V o l .
3 2 ,
N o .
3
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DE T E CT I ON
O F M I CR OS PO RI D I A
BY
IFAT 6 0 9
f r o m
f e m a l e B A L B / c
m i c e
( C h a r l e s R i v e r
L a b o r a t o r i e s ,
W i l m -
i n g t o n ,
M a s s . )
i m m u n i z e d i n t r a p e r i t o n e a l l y
w i t h
f o u r i n o c u l a -
t i o n s o f 5
x
1 0 7
s p o r e s
o f
E .
h e l l e m ,
E .
c l i n i c u t l i ,
o r N .
c o r n e u m
a t 4 - w e e k i n t e r v a l s . S e r a w e r e
c o l l e c t e d
b y t a i l b l e e d i n g
w e e k
a f t e r e a c h i n o c u l a t i o n , p o o l e d
f r o m m i c e
w i t h i n
e a c h
g r o u p ,
a n d
s t o r e d
a t
-
7 0 ° C
u n t i l
u s e d .
S e r a
f r o m t h e
t h i r d
b l e e d i n g
w e r e u s e d
i n t h e s e
s t u d i e s ,
a n d
t h e
ELISA
t i t e r s o b t a i n e d
b y
a s s a y i n g
t h e
s e r a
a g a i n s t
e a c h
h o m o l o g o u s
m i c r o s p o r i d i a n
w e r e
- 1 : 1 2 , 8 0 0 .
T h e
E .
l i e l l e m - i m m u n i z e d B A L B / c
m i c e u s e d f o r
o b t a i n i n g
t h e
p o l y c l o n a l
a n t i s e r a
w e r e t h e n e m p l o y e d
f o r
p r o d u c i n g
M A b s . F o u r
d a y s
a f t e r
t h e f o u r t h
i n o c u l a t i o n , s p l e e n
c e l l s
w e r e
f u s ed w i t h t h e
m u r i n e m y e l o m a
c e l l l i n e
X 6 3 . A g 8 . 6 5 3 ( 1 7 ) .
A p p r o x i m a t e l y 2
w e e k s
l a t e r ,
a n d a f t e r a t
l e a s t
t h r e e medium
c h a n g e s ,
t h e
s u p e r n a t a n t s
w e r e s c r e en e d
f o r
a n t i b o d y p r o d u c -
t i o n
b y
ELISA
b y u s i n g
i n t a c t E .
h e l l e m s p o r e s
a s
a n t i g e n s .
P o s i t i v e h y b r i d o m a s
w e r e
c l o n e d t w i c e
b y
l i m i t i n g
d i l u t i o n s
a n d
s e e d e d
a t
1 0 5
c e l l s p e r
m l o f
c o m p l e t e
RPMI
( c o n t a i n i n g
10
f e t a l
b o v i n e
s e r u m )
i n t o
2 5 - c m 2
t i s s u e c u l t u r e f l a s k s .
T h r e e - d a y
l o g - p h a s e
c u l t u r e
s u p e r n a t a n t s
w e r e u s e d
i n t h e
a s s a y s
d e s c r i b e d
h e r e . T h e
M A b
i s o t y p e s
w e r e
d e t e r m i n e d
w i t h t h e S i g m a
i m m u n o t y p e
k i t
b y f o l l o w i n g
t h e
d i r e c t i o n s
e n c l o s e d ( c a t a l o g
n o .
I S O - 1 ;
S i g m a ,
S t .
L o u i s , M o . ) .
A s c i t e s
f l u i d
w a s p r o d u c e d i n BALB/c
m i c e
b y
i n t r a p e r i t o -
n e a l
i n j e c t i o n o f 0 . 5
m l o f
p r i s t a n e ( 2 , 6 , 1 0 , 1 4 - t e t r a m e t h y l d e -
c a n o i c
a c i d )
f o l l o w e d
b y i n t r a p e r i t o n e a l i n j e c t i o n
o f
1 0
h y b r i -
do ma c e l l s 2 w e e k s
l a t e r ( 1 7 ) . T h e
a s c i t e s
f l u i d w a s
t a p p e d
a p p r o x i m a t e l y
3
w e e k s
l a t e r , c e n t r i f u g e d ( 4 0 0
x
g
f o r
1 5
m i n )
t o
r e m o v e c e l l s ,
a l i q u o t e d ,
a n d s t o r e d a t
-
7 0 ° C
u n t i l u s e d .
E L I S A .
To
p e r f o r m
t h e
E L I S A ,
E .
h e l l e m ,
E .
c u n i c u l i ,
a n d
N .
c o r n e u m
i n
0 . 2
M b i c a r b o n a t e b u f f e r
( p H 9 . 6 )
w e r e
u s e d
t o
c o a t
9 6 - w e l l ,
f l a t - b o t t o m
p o l y s t y r e n e
m i c r o t i t e r
p l a t e s ( C o r n -
i n g G l a s s W o r k s , C o rn i ng , N . Y. )
a t a c o n c e n t r a t i o n o f
5
x
1 0 7
s p o r e s p e r
w e l l a s
d e s c r i b e d
b y
H o l l i s t e r
a n d
C a n n i n g
( 1 8 ) .
MDCK
c e l l
p r o t e i n s
were
a d j u s t e d
t o 1 . 0
m g / m l
i n b i c a r b o n a t e
b u f f e r .
P l a t e s
w e r e
i n c u b a t e d o v e r n i g h t
a t
3 7 ° C ,
w a s h e d w i t h
TBS t h r e e
t i m e s ,
a n d
b l o c k e d
w i t h 3
( w t / v o l )
b o v i n e s e r u m
a l b u m i n
( B S A )
i n TBS
f o r 2
h a t
3 7 ° C .
A f t e r t h r e e
w a s h i n g s
w i t h
T B S ,
1 0 0
R I
o f
t h e
a p p r o p r i a t e
u n d i l u t e d
h y b r i d o m a
o r
c o n t r o l
s u p e r n a t a n t
was a d d e d
t o e a c h w e l l . T h e
p l a t e s
w e r e
i n c u b a t e d
f o r
2
h a t
3 7 ° C ,
w a s h e d t h r e e t i m e s i n
T B S - T w e e n ,
a n d
i n c u b a t e d
f o r
1
h
a t
3 7 ° C
w i t h
a l k a l i n e
p h o s p h a t a s e -
c o n j u g a t e d
g o a t
a n t i - m o u s e
i m m u n o g l o b u l i n
G
( I g G ) - I g M -
I g A
( S i g m a )
d i l u t e d 1 : 4 , 0 0 0
i n TBS
c o n t a i n i n g
1
( w t / v o l )
BS A
( T B S - B S A ) .
A f t e r t h e
p l a t e s
w e r e
w a s h e d
t h r e e t i m e s
w i t h
T B S ,
1 0 0
p . 1
o f t h e e n z y m e s u b s t r a t e
p - n i t r o p h e n y l p h o s -
p h a t e ( 1 . 0 m g / m l
o f d i e t h a n o l a m i n e
b u f f e r , pH
9 . 6 ) w a s
a d d e d ,
a n d
t h e
o p t i c a l
d e n s i t i e s
w e r e
r e a d a t
4 0 5
nm on a n ELISA
s p e c t r o p h o t o m e t e r ( D y n a t e k
L a b o r a t o r i e s
I n c . ,
C h a n t i l l y ,
V a . )
( 1 7 ) .
SDS-PAGE
a n d W e s t e r n
b l o t t i n g
( i m m u n o b l o t t i n g ) .
P a r a -
s i t e
p r o t e i n s
w e r e
s e p a r a t e d b y
s o d i u m
d o d e c y l s u l f a t e - p o l y -
a c r y l a m i d e g e l e l e c t r o p h o r e s i s ( S D S - P A G E ) b y
t h e m e t h o d
o f
L a e m m l i
( 2 0 ) ,
u s i n g
a
5
s t a c k i n g
g e l ,
a
1 0
t o
20
g r a d i e n t
r e s o l v i n g g e l ,
a n d
d i a l l y t a r t a r d i a m i d e
a s t h e
c r o s s - l i n k e r .
P a r -
a s i t e s i n
s a m p l e
b u f f e r
c o n t a i n i n g r 3 - m e r c a p t o e t h a n o l
w e r e
b o i l e d f o r 5 m i n
a n d
c e n t r i f u g e d
a t
1 4 , 0 0 0
x
g f o r
1
m i n t o
r e m o v e
p a r t i c u l a t e
m a t e r i a l s .
E a c h
p r e p a r a t i v e
s l a b
g e l
( 1 6
b y
2 0
c m )
w a s l o a d e d w i t h
2 x
1 0 9
p a r a s i t e s . A f t e r e l e c t r o p h o r e -
s i s ,
t h e
s e p a r a t e d
p o l y p e p t i d e s
w e r e
e l e c t r o p h o r e t i c a l l y t r a n s -
f e r r e d o n t o I m m o b i l o n - P
m e m b r a n e s
( M i l l i p o r e , B e d f o r d ,
M a s s . )
a n d c u t i n t o
s t r i p s .
T h e
s t r i p s
w e r e
i n c u b a t e d
w i t h
5
( w t / v o l )
n o n f a t
d r y
m i l k
( C a r n a t i o n )
i n TBS f o r 3 0 m i n
t o
b l o c k
u n b o u n d
s i t e s , w a s h e d
i n
TBS-Tween f o r 1 0
m i n ,
a n d i n c u -
b a t e d w i t h v a ri o us
d i l u t i o n s
o f
e a c h
h y b r i d o m a s u p e r n a t a n t ,
p o s i t i v e
c o n t r o l
s e r u m ,
o r
n e g a t i v e
c o n t r o l
h y b r i d o m a s u p e r -
n a t a n t .
A f t e r o v e r n i g h t
i n c u b a t i o n t h e s t r i p s w e r e w a s h e d
t h r e e t i m e s w i t h T B S - T w e e n a n d i n c u b a t e d
w i t h a l k a l i n e
p h o s p h a t a s e - c o n j u g a t e d g o a t
a n t i - m o u s e
I g G - l g M ( S i g m a )
d i l u t e d
1 : 1 , 0 0 0
i n
T B S - B S A .
A f t e r i n c u b a t i o n f o r 6 0
m i n
a t
3 7 ° C ,
t h e
s t r i p s
w e r e
w a s h e d
w i t h
t h r e e
c h a n g e s
o f TBS a n d
r i n s e d
w i t h
d e v e l o p i n g
b u f f e r
( 5 0
mM
T r i s - H C l
[ p H
9 . 6 ]
c o n t a i n i n g
3 mM
M g C l , )
a s d e s c r i b e d
p r e v i o u s l y ( 1 ) .
B a n d s
w e r e
v i s u a l i z e d
b y
t h e
a d d i t i o n
o f
t h e s u b s t r a t e s
5 - b r o m o - 4 -
c h l o r o - 3 - i n d o l y l p h o s p h a t e
( 0 . 0 5
m g / m l )
a n d
n i t r o b l u e
t e t r a z o -
l i u m
( 0 . 0 1 m g / m l )
t o
t h e
d e v e l o p i n g
b u f f e r .
A f t e r
c o l o r
d e v e l -
o p m e n t
f o r
3 0
m i n ,
t h e
s t r i p s
w e r e
r i n s e d
i n d i s t i l l e d
w a t e r ,
d r i e d ,
a n d s t o r e d i n t h e d a r k .
I F A T . T h e IFAT
w a s
p e r f o r m e d
a s d e s c r i b e d
p r e v i o u s l y
( 1 7 ) .
T h i n
s m e a r s w e r e
p r e p a r e d
o n
m i c r o s c o p e
s l i d e s
f r o m
i )
p a t i e n t
s t o o l s
c o n t a i n i n g
E .
b i e n e u s i f i x e d w i t h b u f fe re d f o r m a -
l i n - s t o o l
( 1 : 3 , v o l / v o l ) ; i i )
u r i n e
s p e c i m e n s
c o n t a i n i n g
S .
i n t e s t i n a l i s ,
w h i c h
w e r e
c e n t r i f u g e d
a t 4 0 0
x
g
f o r 1 0
m i n
a n d
r e s u s p e n d e d
i n 1 / 5 0
o f
t h e
o r i g i n a l
v o l u m e
o f b u f f er ed f o r m a -
l i n ;
i i i )
f o r m a l i n - f i x e d t i s s u e c u l t u r e - d e r i v e d E .
h e l l e m ,
E .
c i u n i c u l i ,
o r
N .
c o r n e u m ;
a n d
i v )
n o n f i x e d
m i c r o s p o r i d i a
d e r i v e d f r o m t i s s u e
c u l t u r e . T h e s l i d e s
w e r e
a i r
d r i e d ,
f i x e d
i n
1 0 0 m e t h a n o l f o r
5
m i n ,
a n d t h e n
i n c u b a t e d
w i t h
M A b s ,
p o l y c l o n a l
a n t i s e r a ,
o r
a
n e g a t i v e h y b r i d o m a s u p e r n a t a n t .
A f t er i n cu b at i o n
i n
a
m o i s t
c h a m b e r
f o r
3 0
m i n
a t
3 7 ° C ,
t h e
s l i d e s
w e r e
w a s h e d
t w i c e
i n
T B S .
F l u o r e s c e i n
i s o t h i o c y a n a t e -
c o n j u g a t e d
g o a t
a n t i - m o u s e
I g G - I g M - I g A ( S i g m a )
was a b -
s o r b e d w i t h
f o r m a l i n - f i x e d s t o o l
s e d i m e n t ,
d i l u t e d
1 : 2 0 0 ,
a n d
a d d e d t o
t h e
s l i d e s f o r i n c u b a t i o n
a t
3 7 ° C
f o r 3 0
m i n .
T h e n t h e
s l i d e s
w e r e
w a s h e d , c o v e r s l i p s
w e r e
a d d e d w i t h
C y t o s e a l
m o u n t i n g
medium
( S t e p h e n s
S c i e n t i f i c , R i v e r d a l e , N . J . ) ,
a n d
t h e s l i d e s w e r e e x a m i n e d
u n d e r
UV
l i g h t
m i c r o s c o p y
( O l y m -
p u s
A H2
m i c r o s c o p e )
w i t h
a n
e x c i t a t i o n
w a v e l e n g t h
o f 3 8 0 t o
4 9 0
n m ,
w h i c h
g e n e r a t e s
a n
o b s e r v a t i o n
l i g h t w a v e l e n g t h e q u a l
t o
o r
g r e a t e r
t h a n
5 1 5
nm .
RESULTS
C h ar a c t e r i z a t i o n o f MAbs
b y
E L I S A .
T h r e e
h y b r i d o m a
c u l t u r e s ,
w h o s e
s u p e r n a t a n t s r e a c t ed m o s t
s t r o n g l y
t o
E .
h e l l e m
i n t h e
E L I S A ,
w e r e
c l o n e d
t w i c e , d e s i g n a t e d
C 1 2 , E 9 ,
a n d
E l 1 ,
a n d f o u n d
t o
s e c r e t e M A bs o f t h e
I g M i s o t y p e .
T h e
M Abs w e r e
t h e n
t e s t e d
a g a i n b y
ELISA
u s i n g
E . h e l l e m
a s w e l l
a s
E .
c u n i c u l i
a n d N .
c o r n e u m a s
a n t i g e n s ( T a b l e 1 ) .
T h e t h r e e
M Abs r e a c t e d
m o s t
s t r o n g l y
a g a i n s t
E . h e l l e m .
C 1 2
a n d E9 a l s o
r e a c t e d
s t r o n g l y
t o
E .
c u n i c u l l i
a n d
l e s s
s t r o n g l y
a g a i n s t
N .
c o r n e u m ,
a
m i c r o s p o r i d i a n
i s o l a t e d
f r o m t h e c o r n e a l s t r o m a
o f
a human
i m m u n o d e f i c i e n c y
v i r u s - s e r o n e g a t i v e
i n d i v i d u a l
9 ,
3 1 ) .
E l I
p r o d u c e d n e a r l y
i d e n t i c a l
l o w l e v e l s o f
r e a c t i v i t y
a g a i n s t
b o t h
E .
c u n i c u l i
a n d
N .
c o r n e u m .
I n
a l l
c a s e s ,
h o w e v e r ,
t h e
MAbs
p r o d u c e d
a t l e a s t
t h r e e f o l d - h i g h e r
l e v e l s o f r e a c t i v -
i t y a g a i n s t
t h e
m i c r o s p o r i d i a
t h a n
a g a i n s t
MDCK
t i s s u e c u l t u r e
h o s t
c e l l
p r o t e i n s
o r
a g a i n s t
a
n e g a t i v e
h y b r i d o m a
c o n t r o l
s u p e r n a t a n t .
C h a r a c t e r i z a t i o n
o f
MAbs
b y
W e s t e r n
b l o t
i m m u n o a s s a y .
W e s t e r n b l o t
i m m u n o d e t e c t i o n
o f
E . h e l l e m
w a s
p e r f o r m e d
t o
d e t e r m i n e
t h e
m o l e c u l a r
w e i g h t s
o f
p r o t e i n s
r e c o g n i z e d
b y
t h e
M A b s .
A l l
t h r e e
I g M
M A bs
d e t e c t e d
p r o t e i n s
w i t h
M r S
o f
6 2 ,
6 0 ,
a n d
5 0
a t t h e
h i g h e s t
d i l u t i o n s
p r o d u c i n g
r e a c t i v i t y .
L e s s
d i l u t e d
h y b r i d o m a s u p e r n a t a n t s
a l s o b o u n d t o
p r o t e i n s
w i t h
M r s
o f
5 5
a n d
5 3
( F i g .
1 ) .
I d e n t i c a l
r e s u l t s
w e r e o b t ai n e d
w h e n
p a r a s i t e p r o t e i n s
w e r e
p r e p a re d b y u s i n g
t h e
p r o t e a s e
i n h i b i -
t o r s
l e u p e p t i n
( 0 . 5 , u g / m l )
a n d
p h e n y l m e t h y l s u l f o n y l
f l u o r i d e
0 .
2
m M ) .
I F A T .
M u r i n e
p o l y c l o n a l
a n t i s e r a t o E .
c u n i c u l i
a n d
E .
h e l l e m ,
a s
w e l l
a s t h e M Abs r a i s e d
a g a i n s t
E .
h e l l e m ,
w e r e
u s e d
t o
d e t e c t
m i c r o s p o r i d i a b y
I F A T . S m e a r s o f
t i s s u e
c u l t u r e -
V O L .
3 2 ,
1 9 9 4
8/20/2019 Detection of Microsporidia by Indirect Immunofluorescence
http://slidepdf.com/reader/full/detection-of-microsporidia-by-indirect-immunofluorescence 3/5
6 1 0
A L D R A S
ET A L .
T A B L E 1 .
ELI SA r e a c t i v i t i e s o f
MAbs r a i s e d t o E .
h e l l e m a
ELISA v a l u e s
f o r a b s o r b a n c e t o a n t i g e n
t a r g e t
M A b
E .
h e l l e m
E .
c u n i c u l i
N . c o r n e u m
MDCK
c e l l s
C 1 2 1 . 1 5 3
±
0 . 0 1 1
0 . 7 8 5
±
0 . 0 9 0
0 . 5 5 5 ±
0 . 0 0 5
0 . 1 0 1 ± 0 . 0 0 6
E 9
0 . 9 5 3
±
0 . 0 5 8
0 . 4 9 1
±
0 . 0 1 4 0 . 3 4 1
± 0 . 0 7 0
0 . 0 9 4 ± 0 . 0 0 2
E l i
0 . 8 1 8
±
0 . 0 7 2
0 . 3 1 0
±
0 . 0 3 3
0 . 3 1 8 ±
0 . 0 4 3
0 . 0 5 2
±
0 . 0 1 3
N e g .
s u p t .
0 . 1 0 4
±
0 . 0 0 7
0 . 0 5 5
±
0 . 0 3 0 0 . 0 2 0
±
0 . 0 2 7
- 0 . 0 4 5
±
0 . 0 0 0 1
a
U n d i l u t e d c u l t u r e s u p e r n a t a n t s
f r o m t w i c e - c l o n e d h y b r i d o m a s
w e r e r e c o v e r e d
a n d a s s a y e d
a s d e s c r i b e d i n M a t e r i a l s a n d M et h o d s .
b
V a l u e s a r e
t h e
m e a n s
o f t w o
r e p l i c a t e s
±
t h e
s t a n d a r d
d e v i a t i o n s .
d e r i v e d E . h e l l e m , E . c u n i c u l i , N .
c o r n e u m , f o r m a l i n - f i x e d
s t o o l s m i x e d w i t h t h e
t i s s u e
c u l t u r e - d e r i v e d
o r g a n i s m s , a n d
s t o o l
a n d
u r i n e s p e c i m e n s
w i t h E .
b i e n e u s i a n d S . i n t e s t i n a l i s ,
r e s p e c t i v e l y ,
w e r e
e x a m i n e d . T h e
m u r i n e
a n t i s e r u m
t o E .
C ,
C . )
t o
.
_
ci
: }
a ) d L
i J
UJ
E
k D a
_
1 0 6
-
8 0
4 9
-
3 2
2 7
1 8
U
F I G .
1 .
W e s t e r n
b l o t c h a r a c t e r i z a t i o n
o f
M A b s t o
E .
h e l l e m .
E .
h e l l e m
p r o t e i n s
w e r e
s e p a r a t e d
b y SDS-PAGE
u n d e r
r e d u c i n g
c o n d i -
t i o n s a n d
t r a n s f e r r e d t o
I m m o b i l o n - P
m e m b r a n e s .
T h e
m e m b r a n e
a n t i g e n
s t r i p s
w e r e i n c u b a t e d
w i t h MAbs
( C 1 2 ,
E 9 ,
a n d
E l i )
h a r v e s t e d
f r o m
3 - d a y l o g - p h a s e h y b r i d o m a
c e l l c u l t u r e s
a n d d i l u t e d
1 : 1 0 ;
t h i s
was
f o l l o w e d
b y
i m m u n o d e t e c t i o n
u s i n g
a l k a l i n e
p h o s p h a t a s e - c o n j u g a t e d
g o a t
a n t i - m o u s e
I g M
a n d c o l o r
d e v e l o p m e n t
w i t h
n i t r o b l u e t e t r a z o -
l i u m
a n d
5 - b r o m o - 4 - c h l o r o - 3 - i n d o l y l p h o s p h a t e .
A s c i t e s
f l u i d
f r o m
B A L B / c
m i c e i n o c u l a t e d
w i t h
h y b r i d o m a
E9 c e l l s
w a s
d i l u t e d
1 : 1 0 0 ,
a n d t h e
h y p e r i m m u n e
m u r i n e
p o l y c l o n a l
a n t i s e r u m
t o E . h e l l e m
( m
a
E .
h e l l e m ) w a s
u s e d
a t a
d i l u t i o n
o f 1 : 2 , 0 0 0 .
M o l e c u l a r
m a s s e s
o f
s t a n d a r d s
a r e
g i v e n
a t t h e
r i g h t
( i n
k i l o d a l t o n s ) .
N e g .
s u p t . , n e g a t i v e
s u p e m a t a n t .
h e l l e m ( d i l u t e d
1 : 5 0 0 )
d e t e c t e d
b o t h
n a t i v e E . h e l l e m ( d a t a
n o t
s h o w n )
a n d f o r m a l i n - f i x e d
E .
h e l l e m
( F i g .
2 A )
a n d
E . c u n i c u l i
( d a t a
n o t
s h o w n ) ,
a s
w e l l
a s E . b i e n e u s i
( F i g . 2 1 3 ) a n d
S .
i n t e s t i n a l i s ( F i g . 2 C )
i n
p a t i e n t
s t o o l s a n d
u r i n e ,
r e s p e c t i v e l y .
T h e
m u r i n e p o l y c l o n a l a n t i s e r u m
t o E . c u n i c u l i
p r o d u c e d
r e s u l t s
i d e n t i c a l t o
t h o s e
o b t a i n e d w i t h t h e
m u r i n e p o l y c l o n a l
a n t i s e r u m t o
E .
h e l l e m ( d a t a
n o t s h o w n ) .
T h e
t h r e e MAbs
p r o d u c i n g
t h e s t r o n g e s t
r e a c t i v i t y t o
E . h e l l e m i n t h e
ELISA
a l s o
d e t e c t e d E . h e l l e m ,
E .
c u n i c u l i ,
E . b i e n e u s i ,
a n d
S .
i n t e s t i -
n a l i s
b u t
n o t N . c o r n e u m
b y I F A T .
R e s u l t s
a r e
s h o w n f o r
E9
d e t e c t i o n o f
E .
h e l l e m
( F i g .
2 D ) ,
E .
b i e n e u s i
( F i g .
2 E ) ,
a n d
S .
i n t e s t i n a l i s ( F i g . 2 F ) .
IFA
s t a i n i n g
o f E .
b i e n e u s i
w i t h t h e
MAbs
( F i g . 2 E )
w a s
n o t
a s s t r o n g
a s
w i t h
t h e p o l y c l o n a l
a n t i s e r a ( F i g .
2 B ) .
T h e MAbs d i d n o t
s t a i n
C r y p t o s p o r i d i u m ,
I s o s p o r a , G i a r -
d i a ,
C a n d i d a ,
o r
T r i c h o m o n a s s p p .
i n k n o w n p o s i t i v e p a t i e n t
s t o o l
s p e c i m e n s
( d a t a
n o t s h o w n ) . T h e
m u r i n e h y p e r i m m u n e
s e r a r a i s e d
a g a i n s t
E .
h e l l e m o r E . c u n i c u l i d i d d e t e c t
N .
c o r n e u m a n d C a n d i d a a l b i c a n s
w h e n u s e d a t d i l u t i o n s
o f
1 : 2 0 0
o r
l o w e r ,
a n d
t h e
n o n s p e c i f i c
b i n d i n g t o y e a s t c e l l s c o u l d
b e
r ed u c ed b y
a b s o r b i n g
w i t h
C .
a l b i c a n s
( d a t a n o t
s h o w n ) .
I n
a d d i t i o n ,
b a c k g r o u n d
s t a i n i n g , p a r t i c u l a r l y
w h e n t h e
p o l y c l o n a l
a n t i s e r a
w e r e
u s e d ,
c o u l d b e f u r t h e r
r e d u c e d b y a b s o r b i n g
t h e
F I G . 2 .
IFAT f o r
d e t e c t i n g m i c r o s p o r i d i a
u s i n g
p o l y c l o n a l
a n t i b o d -
i e s
a n d
M A b s . F o r m a l i n - f i x e d
t i s s u e
c u l t u r e - d e r i v e d
E .
h e l l e m
( A
a n d
D ) ,
E .
b i e n e u s i i n s t o o l ( B
a n d
E ) ,
a n d S .
i n t e s t i n a l i s
i n
u r i n e
( C
a n d
F )
w e r e
s t a i n e d w i t h
m u r i n e
h y p e r i m m u n e
p o l y c l o n a l
a n t i s e r u m r a i s e d
a g a i n s t
E .
h e l l e m
( A
t o
C )
o r
M b E9
( D
t o
F )
a s
d e s c r i b e d
i n
M a t e r i a l s
a n d M e t h o d s .
J . C L I N .
M I C R O B I O L .
8/20/2019 Detection of Microsporidia by Indirect Immunofluorescence
http://slidepdf.com/reader/full/detection-of-microsporidia-by-indirect-immunofluorescence 4/5
DETECTION
OF
MICROSPORIDIA
BY
IFAT 6 1 1
f l u o r e s c e i n
i s o t h i o c y a n a t e - c o n j u g a t e d
a n t i b o d i e s w i t h
f o r m a -
l i n - f i x e d
s t o o l
s e d i m e n t .
DISCUSSION
O p p o r t u n i s t i c
i n f e c t i o n s w i t h m i c r o s p o r i d i a
i n
AIDS
p a -
t i e n t s
a r e i n c r e a s i n g l y
b e i n g
r e p o r t e d ,
y e t t h e
n u m b e r o f
m i c r o s p o r i d i o s i s
c a s e s
i s p r o b a b l y
g r e a t l y
u n d e r r e p o r t e d .
M i -
c r o s p o r i d i u m
i n f e c t i o n s a r e d i f f i c u l t t o d i a g n o s e ,
p r i m a r i l y
b e c a u s e t h e o r g a n i s m s
a r e
s m a l l a n d d i f f i c u l t
t o d i s t i n g u i s h
f r o m
b a c t e r i a
a n d
s m a l l y e a s t s i n t i s s u e a n d
s t o o l . G i e m s a
s t a i n
( 2 5 ,
3 2 )
a n d a
m o d i f i e d
t r i c h r o m e
s t a i n u s i n g
c h r o m o t r o p e
2R
( 3 5 )
h a v e b e e n
u s e d
t o d e t e c t
m i c r o s p o r i d i a
i n
s t o o l , b u t
w i t h
s o m e d i f f i c u l t i e s .
G i e ms a - s t a i ne d m i cr o s p o r i d i a
a r e
b l u e
a n d
d i s p l a y
a
p u r p l e - b l u e
n u c l e u s
w h i c h d i s t i n g u i s h e s
t h e m
f r o m
b a c t e r i a . I t i s d i f f i c u l t ,
h o w e v e r , t o
f i n d
m i c r o s p o r i d i a i n s t o o l
s m e a r s
i n w h i c h m o s t o t h er o r g a n i s m s
a l s o s t a i n
b l u e .
T h e
m o d i f i e d t r i c h r o m e ( c h r o m o t r o p e
2 R ) s t a i n i n g
m e t h o d d e -
s c r i b e d b y
W e b e r e t
a l .
( 3 5 )
h a s t h e a d v a n t a g e
t h a t
m o s t
b a c t e r i a
c o u n t e r s t a i n l i g h t
g r e e n , l e a v i n g t h e
m i c r o s p o r i d i a
p i n k .
T h e s t o o l
s m e a r m u s t b e
v e r y
t h i n i n
o r d e r
t o o b s e r v e t h e
i n t e r n a l
s t r u c t u r e
o f t h e
m i c r o s p o r i d i a ,
s o m i c r o s p o r i d i a
may
b e m i s s e d
i f
t h e p a r a s i t e
b u r d e n i s l o w
o r
i f
m i c r o s p o r i d i a
a r e
m i x e d
w i t h m u cu s .
I n
a d d i t i o n ,
s m a l l
y e a s t s
a n d
s o m e
b a c t e r i a
i n s t o o l a l s o s t a i n p i n k ,
w h i c h c a n
c o m p l i c a t e t h e r e s u l t s .
F i n a l l y , i t
i s c r u c i a l
t o
m o n i t o r
t h e d e s t a i n i n g
s t e p s o t h a t
t h e
m i c r o s p o r i d i a
r e m a i n p i n k
b u t h i g h
b a c k g r o u n d l e v e l s o f
s t a i n i n g
a r e n o t
g e n e r a t e d ( 1 6 ) .
A c a l c o f l u o r
s t a i n i n g
m e t h o d
u t i l i z i n g
U v e t i x
2 B
( C i b a - G e i g y )
o r
f l u o r e s c e n t
b r i g h t e n e r
( c a t a l o g
n o .
F - 6 2 5 9 ;
S i g m a )
a l s o may b e u s e f u l f o r
d e t e c t i n g
m i c r o s p o r i d i a ( 3 3 ) .
T h e m i c r o s p o r i d i a
d i s p l a y
a r e l a t i v e l y t h i c k
r i n g
o f f l u o r e s c e n c e ,
a n d t h e
a n t e r i o r
r e g i o n a p p e a r s
c o n c a v e .
H o w e v e r ,
b e c a u s e
y e a s t s
a l s o s t a i n
w i t h c a l c o f l u o r , f u r t h e r
s t u d i e s
a r e n e e d e d
t o a s s e s s t h e s p e c i f i c i t y
a n d
r e l i a b i l i t y
o f
t h e s e
s t a i n s .
T h e u s e
o f
m i c r o s p o r i d i u m - s p e c i f i c
a n t i b o d i e s
i n IFAT
p r o c e d u r e s a p p e a r s
t o o v e r c o m e
s o m e o f t h e s e
d i f f i c u l t i e s .
I n
e a r l i e r s t u d i e s ,
IFAT
p r o c e d u r e s
w i t h
p o l y c l o n a l
a n t i s e r a w e r e
u s e d
t o
s h o w
t h a t
s e v e r a l s p e c i e s
o f m i c r o s p o r i d i a
d e m o n -
s t r a t e d
i m m u n o l o g i c a l
c r o s s - r e a c t i v i t y ( 2 1 ) .
More
r e c e n t l y ,
p o l y c l o n a l
a n t i s e r a
p r o d u c e d
a g a i n s t
E . c u n i c u l i
a n d
E . h e l l e m
w e r e u s e d t o d i a g n o s e
o c u l a r a n d
s y s t e m i c E . h e l l e m
i n f e c t i o n s
( 2 7 ,
2 8 ) ,
a n d
p o l y c l o n a l
a n t i s e r u m r a i s e d a g a i n s t
E . c u n i c u l i
i n
r a b b i t s
w a s u s e d
i n t h e IFAT t o
d e t e c t
E . b i e n e u s i o r g a n i s m s i n
d e p a r a f f i n i z e d
t i s s u e
s e c t i o n s
( 3 6 )
a n d
i n
s t o o l ( 3 7 ) .
I n
t h i s
s t u d y ,
we
o b s e r v e d t h a t
p o l y c l o n a l
a n t i s e r a r a i s e d
a g a i n s t
E . c u n i c u l i
a n d E . h e l l e m i n
m i c e ,
a s w e l l a s
t h e
t h r e e
M A b s ,
d e t e c t e d
f o r m a l i n - f i x e d ,
t i s s u e
c u l t u r e - d e r i v e d E n c e p h -
a l i t o z o o n
s p e c i e s
a s
w e l l
a s E . b i e n e u s i i n f o r m a l i n - f i x e d s t o o l .
T h e
p o l y c l o n a l a n t i s e r a
t o
E .
c u n i c u l i
a n d E .
h e l l e m
a l s o
d e t e c t e d
a
n e w l y
d e s c r i b e d m i c r o s p o r i d i a n ,
S .
i n t e s t i n a l i s
( 3 ,
2 4 ) ,
i n f o r m a l i n - f i x e d u r i n e .
T h e a n t i s e r u m r a i s e d a g a i n s t
E .
c u n i c u l i
a n d
E . h e l l em d e t ec t ed
N .
c o r n e u m
o n l y
w h e n
u s e d a t
l o w e r
d i l u t i o n s ,
s u g g e s t i n g
t h a t
E .
h e l l e m
a n d
E .
c u n i c u l i
a r e
m o r e
c l o s e l y
r e l a t e d
t o E .
b i e n e u s i
a n d
S .
i n t e s t i n a l i s
t h a n
t o
N .
c o rn e u m. U nl i k e
t h e
p o l y c l o n a l
a n t i s e r a r a i s e d a g a i n s t
E .
c u n i c u l i
a n d
E .
h e l l e m ,
t h e
h y b r i d o m a
s u p e r n a t a n t s
d i d n o t
s t a i n
N .
c o r n e u m
o r
y e a s t
c e l l s
i n
t h e I F A T , e v e n
t h o u g h
t h e y
d i d r e a c t s o m e w h a t t o N . c o r n e u m i n t h e E L I S A . M i c r o s p o -
r i d i a
o f t h e g e n u s Nosema
t y p i c a l l y i n f e c t
i n s e c t s , a n d
t h e
l a c k
o f c r o s s - r e a c t i v i t y
i n t h e
IFAT
i s
m o s t
l i k e l y
d u e t o a l o w e r
d e g r e e
o f
p h y l o g e n e t i c
r e l a t e d n e s s ( 3 4 ) .
T h e M A bs
a n d
p o l y c l o n a l
a n t i b o d i e s p r o v i d e d
d i f f e r e n t
a d v a n t a g e s
i n
t h e
IFA s t a i n .
E .
b i e n e u s i s t a i n e d
m o r e i n t e n s e l y
w i t h
t h e
p o l y c l o n a l
a n t i s e r a
r a i s e d a g a i n s t E .
c u n i c u l i o r
E .
h e l l e m
t h a n w i t h
t h e
MAbs
r a i s e d a g a i n s t E . h e l l e m .
H o w e v e r ,
we
f o u n d t h a t t h e p o l y c l o n a l
a n t i s e r a g e n e r a t e d m o r e b a c k -
g r o u n d
i n f o r m a l i n - f i x e d s t o o l
s p e c i m e n s w i t h t h e
IFA
s t a i n .
T h e
d e g r e e o f
b a c k g r o u n d d e p e n d e d
o n t h e d i l u t i o n o f
a n t i -
s e r u m ,
a s a l s o d e s c r i b e d
b y
W e i s s
e t a l . ( 3 6 ) .
We a l s o f o u n d
t h a t t h e p o l y c l o n a l
a n t i s e r a
s t a i n e d
y e a s t c e l l s , b u t
t h i s p r o b l e m
c o u l d
b e o v e r c o m e
b y
a b s o r b i n g
t h e
a n t i s e r a w i t h C .
a l b i c a n s .
N e i t h e r
t h e C a n d i d a - a b s o r b e d
p o l y c l o n a l a n t i s e r a
n o r
t h e
MAbs
s t a i n e d y e a s t
c e l l s ,
b a c t e r i a , o r
C r y p t o s p o r i d i u m ,
I s o -
s p o r a , G i a r d i a ,
o r
T r i c h o m o n a s
s p p . ;
b o t h t h e a n t i s e r a
a n d
MAbs
t h u s a p p e a r e d
t o
b e
s p e c i f i c
f o r
m i c r o s p o r i d i a .
I n
a d d i t i o n ,
we f o u n d t h a t a b s o r b i n g
t h e
f l u o r e s c e i n
i s o t h i o c y a -
n a t e - c o n j u g a t e d a n t i b o d y
w i t h
s e d i m e n t e d
s t o o l m a t e r i a l
h e l p e d r e d u c e
t h e b a c k g r o u n d
s t a i n i n g .
T h a t t h e m o s t
s t r o n g l y r e a c t i n g
MAbs
s e l e c t e d f o r
c l o n i n g
w e r e
o f
t h e I g M i s o t y p e
w a s p r o b a b l y
d u e
t o
t h e
p e n t a m e r i c
s t r u c t u r e
o f t h e
IgM a n t i b o d i e s ,
w h i c h c o u l d a m p l i f y
t h e
s i g n a l
i n t h e
ELISA
m o r e
t h a n m o n o m e r i c
I g G
M A bs c o u l d .
A l -
t h o u g h t h e t h r e e
MAbs a p p e a r e d
t o
d e t e c t t h e
s a m e p r o t e i n s
o n W e s t e r n
b l o t s
o f
E . h e l l e m ,
b i n d i n g b y
E9 a n d
E 1 i ( b u t
n o t
C 1 2 )
w a s
l o s t
b y
p e r i o d a t e
o x i d a t i o n ,
s u g g e s t i n g
t h a t
t h e
e p i t o p e s r e c o g n i z e d
b y
E9 a n d
E l 1 a r e c a r b o h y d r a t e s
( u n p u b -
l i s h e d o b s e r v a t i o n s ) .
I n
a d d i t i o n ,
t h e
ELISA
v a l u e s
f o r a b s o r -
b a n c e b y t h e MAbs
a g a i n s t t h e
d i f f e r e n t
m i c r o s p o r i d i a
v a r i e d ,
s o
t h a t
i t
i s
p o s s i b l e
t h a t
t h e
MAbs
d e t e c t e d
d i f f e r e n t
e p i t o p e s
o n t h e s a m e p r o t e i n s .
T h e
f a c t
t h a t s e v e r a l
p r o t e i n s w e r e
d e t e c t e d
b y
e a c h
M A b c o u l d
b e d u e
t o t h e
p r e s e n c e
o f
t h e
s a m e
e p i t o p e s
o n p r e c u r s o r
m o l e c u l e s a n d / o r b r e a k d o w n
p r o d u c t s
i n
t h e p a r a s i t e
p r e p a r a t i o n s u s e d
t o
p r e p a r e
t h e b l o t s .
A d d i n g t h e p r o t e a s e i n h i b i t o r s l e u p e p t i n
a n d p h e n y l m e t h y l s u l -
f o n y l f l u o r i d e
t o t h e p a r a s i t e
p r e p a r a t i o n s ,
h o w e v e r , s t i l l
r e s u l t e d i n d e t e c t i o n
o f
t h e m u l t i p l e b a n d s .
F i n a l l y ,
i t
i s
p o s s i b l e
t h a t t h e s a m e e p i t o p e
i s f o u n d
o n
f u n c t i o n a l l y
d i f f e r -
e n t p r o t e i n s .
T h e
a v a i l a b i l i t y
o f
p o l y c l o n a l
a n t i b o d i e s
t o E n c e p h a l i t o z o o n
s p e c i e s
a n d
MAbs
t o E .
h e l l e m
s h o u l d b e
p a r t i c u l a r l y u s e f u l
i n
s c r e e n i n g
s p e c i m e n s
f o r t h e p r e s e nc e o f
t h e m o s t
common
m i c r o s p o r i d i a
c a u s i n g
o p p o r t u n i s t i c
i n f e c t i o n s
i n AIDS p a -
t i e n t s .
A d d i t i o n a l s p e c i m e n s
n e e d
t o b e s t a i n e d w i t h
t h e s e
r e a g e n t s
t o
d e t e r m i n e
t h e i r
f e a s i b i l i t y
a n d
r e l i a b i l i t y
f o r
r o u -
t i n e d i a g n o s t i c
a n d
e p i d e m i o l o g i c
s t u d i e s .
I n a d d i t i o n , I g G
MAbs w h i c h ,
i n c o m b i n a t i o n , may p r o v e m o r e
u s e f u l t h a n IgM
MAbs a r e b e i n g
e v a l u a t e d . M o s t o f
t h e MAbs
g e n e r a t e d
a g a i n s t E .
h e l l e m w e r e o f
t h e
IgM
i s o t y p e , a n d
t h e s e MAbs
r e a c t e d m o r e i n t e n s e l y
t h a n t h e I g G
M A b s . H o w e v e r ,
we
a r e
f i n d i n g
t h a t
t h e
u s e o f t h e
IgM MAbs c h a r a c t e r i z e d
i n t h i s
s t u d y may
n o t
b e f e a s i b l e f o r
ELISA p r o c e d u r e s
b e c a u s e o f
h i g h - l e v e l , n o n s p e c i f i c
r e a c t i v i t y . T h i s
may b e d u e t o t h e h i g h e r
l e v e l o f
s e n s i t i v i t y i n ELISA t h a n
i n
IFA
s t a i n i n g .
A n o t h e r
d i f f i c u l t y
i s t h a t t h e s e c o n d a r y
o r c o n j u g a t e d a n t i b o d i e s
n e e d t o
b e a b s o r b e d
w i t h
s e d i m e n t e d s t o o l t o r e d u c e
b a c k g r o u n d
i n
t h e I F A T ,
a n d i t
may
n o t
b e
p o s s i b l e
t o r e m o v e t h i s
i n t e r f e r -
e n c e
f o r
t h e
E L I S A .
To
r e d u c e t h i s
b a c k g r o u n d i n t e r f e r e n c e ,
o n e c o u l d
c o n j u g a t e
t h e MAb
e . g . , w i t h
f l u o r e s c e i n
i s o t h i o -
c y a n a t e
o r a l k a l i n e p h o s p h a t a s e ) ,
w h i c h
i s e a s i e r
w i t h
I g G t h a n
w i t h
1 g M
M A b s . T h e
p o l y c l o n a l
a n t i b o d i e s
a n d
M A bs
d e -
s c r i b e d
i n t h i s s t u d y , h o w e v e r ,
p r o v i d e
a b a s i s f o r s c r e e n i n g
s p e c i m e n s
f o r
t h e p r e s e n c e
o f m i c r o s p o r i d i a
b y
IFA s t a i n i n g .
S p e c i e s - s p e c i f i c
d i a g n o s i s
o f
m i c r o s p o r i d i u m - p o s i t i v e
s p e c i -
mens s h o u l d t h e n b e c o m e
p o s s i b l e
a s s p e c i e s - s p e c i f i c
i m m u -
n o l o g i c
a n d m o l e c u l a r p r o b e s b e c o m e
a v a i l a b l e .
ACKNOWLEDGMENTS
We t h a n k Donna B e r t u c c i f o r
e x c e l l e n t
t e c h n i c a l
a s s i s t a n c e
a n d
M u r p h y
Dowous
f o r
p h o t o g r a p h i c
a s s i s t a n c e .
T h i s s t u d y w a s
s u p po r t ed b y
g r a n t s EY08778 a n d
R R 0 0 1 6 4 f r o m
t h e
N a t i o n a l I n s t i t u t e s o f H ea l t h.
V O L .
3 2 ,
1 9 9 4
8/20/2019 Detection of Microsporidia by Indirect Immunofluorescence
http://slidepdf.com/reader/full/detection-of-microsporidia-by-indirect-immunofluorescence 5/5
6 1 2 A L D R A S
E T A L .
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h e l l e m
( n . s p . ) ,
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u n d e s c r i b e d
i n
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e n -
t e r o c y t e s
a n d
m a c r o p h a g e s ,
a n d
a s s o c i a t e d w i t h
d i a r r h e a
i n a n
a c q u i r e d
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Hum.
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o f
s p o r e s
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E n t e r o c y t o z o o n
b i e n e u s i
i n
s t o o l
a n d
d u o d e n a l
f l u i d f r o m
AI DS
p a t i e n t s .
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h e l l e m )
a n d
a c q u i r e d
i m m u n o d e f i c i e n c y
s y n d r o m e .
A r c h .
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D i e s e n h o u s e ,
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W e b e r ,
R .
L .
F o n t ,
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W i l s o n ,
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C o r r e n t ,
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N .
S e r a d a r e v i c ,
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R o s b e r g e r ,
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K e e n e n ,
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( E n c e p h a l i t o -
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w h o l e - c e l l
a n t i s e r a
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