SEROLOGICAL DIFFERENTIATION OF MICROSPORIDIA
WITH SPECIAL REFERENCE TO TRACHIPLEISTOPHORA HOMINIS
C H E N E Y S.A.*, L A F R A N C H I - T R I S T E M N.J.* & C A N N I N G E . U . *
S u m m a r y :
Myositis is a common clinical syndrome in advanced stages of AIDS. Trachipleistophora hominis (phylum Microspora) has been detected in several cases of painful, immobilising myositis in AIDS patients. Enzyme linked immunosorbent assays (ELISAs) and Western blotting of protein profiles separated by SDS PAGE were used to determine whether this species could be detected and differentiated by serology. Sixteen microsporidia, including severa species known to infect man and species infecting fish,
crustaceans and a mosquito, were used as antigen. Each species had a unique profile of SDS PAGE-separated proteins. In Western blots, mouse antiserum, raised to T. hominis and selected for its high ELISA specificity, bound to antigens ranging from less than 25 kDa to greater than 2 5 0 kDa with major bands at 39-44 kDa and 98-f 5 0 kDa on T. hominis protein profiles. The serum also recognised some high molecular weight antigens in the profiles of Vavraia culicis, Heterosporis anguillarum, and three species of Pleistophora but none in the remaining genera examined. It was concluded that ELISA and Western blotting could be used to detect and differentiate T. hominis in muscle biopsy tissue from patients with myositis. However, sera from T. hominis-infected patients in the terminal stages of AIDS would not be useful for detection of infections because of a sharp decline in antibody level.
KEYWORDS : Trachipleistophora hominis, microsporidia, human myositis, fish, crustaceans, mosquitoes, ELISA, SDS PAGE, Western blot.
I N T R O D U C T I O N
T
h e m i c r o s p o r i d i a n parasite Trachipleistophora hominis H o l l i s t e r , C a n n i n g , W e i d n e r , F i e l d , K e n c h & Marriott, 1 9 9 6 w a s found infecting the s k e l e t a l m u s c l e a n d c o r n e a a n d w a s p r e s e n t in nasal d i s c h a r g e o f an AIDS patient (Field et al., 1 9 9 6 ) . T h e patient h a d b e c o m e i m m o b i l i s e d b y s e v e r e m u s c u l a r pain and wasting but his condition improved after treat- m e n t with a t h e r a p e u t i c c o m b i n a t i o n w h i c h i n c l u d e d a l b e n d a z o l e ( 4 0 0 mg t w i c e daily), a drug with k n o w n a n t i - m i c r o s p o r i d i a l a c t i v i t y . T h e p a t i e n t d i e d five* Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2AZ, U.K.
Correspondence: Elizabeth U. Canning, Imperial College at Silwood Park, Ascot, Berks SL5 7PY, U.K.
Tel.: 020 7594 2244 - Fax: 020 7594 2339.
E-mail: [email protected]
Résumé : DIFFÉRENCIATION SÉROLOGIQUE DES MICROSPORIDIES ET NOTAMMENT DE TRACHIPLEISTOPHORA HOMINIS
La myosite est un syndrome fréquent des stades avancés du SIDA.
Trachipleistophora hominis (phylum Microspora) a éfé détecté dans plusieurs cas de myosite douloureux, immobilisant les patients atteints du SIDA. L'ELISA et le Western blot des protéines séparées par SDS PAGE ont été utilisés pour déterminer si cette espèce peut être détectée et différenciée par sérologie. Outre T . hominis, quinze autres espèces trouvées respectivement chez l'homme, des poissons, des crustacés ou des moustiques ont été utilisées comme matériel antigénique. Le profil des protéines séparées par le SDS PAGE était propre à chaque espèce. En Western blot, un sérum de souris dirigé contre T . hominis et choisi pour sa haute spécificité en ELISA s'est lié à des antigènes compris entre
< 25 kDa et > 250 kDa avec des bandes principales à 39- 44 kDa et 98-150 kDa dans les profils de protéines obtenus à partir de T . hominis. Ce même sérum a également reconnu des antigènes de haut poids moléculaire de Vavraia culicis, d'Heterosporis anguillarum et de trois espèces de Pleistophora, mais aucun dans les autres genres testés. En conclusion, l'ELISA et le Western blot peuvent être utilisés pour détecter et différencier T. hominis dans les biopsies musculaires des patients atteints de la myosite. Toutefois, le sérum des patients contaminés par cette microsporidie aux stades terminaux du SIDA serait inutilisable pour sa détection en raison de la chute du taux des anticorps sériques.
MOTS CLÉS : Trachipleistophora hominis, microsporidies, myosite humaine, poisson, crustacé, moustiques, ELISA, SDS PAGE, Western blot.
m o n t h s after a d m i s s i o n to hospital o f p r o g r e s s i v e HIV d i s e a s e without r e c u r r e n c e o f myositis. T h e m i c r o - sporidia w e r e isolated in vitro a n d d e s c r i b e d as a n e w g e n u s a n d s p e c i e s (Hollister et al., 1 9 9 6 ) .
In n u m e r o u s g e n e r a o f m i c r o s p o r i d i a , s p o r o g o n y cul- m i n a t e s in a large ( m o r e t h a n e i g h t ) a n d variable n u m b e r o f s p o r e s p a c k a g e d in an e n v e l o p e o f pre- s u m e d parasite origin ( p o l y s p o r o u s in s p o r o p h o r o u s v e s i c l e s ) . O f t h e s e o n l y Trachipleistophora from m a n and Glugea, Heterosporis and Pleistophora from fish are k n o w n from vertebrates Pleistophora-like microsporidia also infect e d i b l e c r u s t a c e a n s - shrimps and c r a b s .
Glugea and Heterosporis infections are strictly localised in their h o s t s b y b e i n g r e t a i n e d within h y p e r t r o p h i e d host cells modified to form x e n o m a s . Pleistophora a n d
Trachipleistophora infections are diffuse. S p e c i e s o f the g e n e r a Loma a n d Spraguea are a l s o parasites o f fish.
In Loma, s p o r o g o n y gives rise to four o r m o r e s p o r e s retained within a p a r a s i t o p h o r o u s v a c u o l e b o u n d e d b y
Parasite, 2001, 8, 91-97
Mémoire 91
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2001082091
C H E N E Y S.A., L A P R A N C H I - T R I S T E M N . J . & C A N N I N G E . U .
a m e m b r a n e o f p r e s u m e d host origin (Lom & P e k k a - rinen, 1 9 9 9 ) . In Spraguea there are t w o s p o r o g o n i c s e q u e n c e s w h i c h give rise to t w o types o f spores, b o t h lying free in host cell cytoplasm ( n o e n v e l o p i n g m e m b r a n e ) .
In t w o c a s e s o f m i c r o s p o r i d i a n m y o s i t i s in AIDS patients, the parasites w e r e identified as Pleistophora spp. ( C h u p p et al, 1 9 9 3 ; Grau et al, 1 9 9 6 ) but are m o r e likely to b e l o n g to Trachipleistophora than Pleis
tophora (Canning, 2 0 0 1 ) and another T. hominis infec
tion has b e e n d i a g n o s e d recently (A. Curry, personal c o m m u n i c a t i o n ) . A s e c o n d s p e c i e s Trachipleistophora anthropophthera has c a u s e d a multisystem dissemina
ting infection, especially infecting the brain, in t w o m o r e AIDS patients ( Y a c h n i s et al., 1 9 9 6 ; Vavra et al, 1 9 9 8 ) . T h e o c c u r r e n c e o f several o f t h e s e d e e p tissue infections, w h i c h c a n n o t b e transmitted directly bet
w e e n h u m a n s , at least not from m u s c l e or brain, has raised the question o f their origin, and ingestion o f infected fish or crustaceans s e e m e d a likely possibi
lity. However, in a recent study o f generic relationships r e v e a l e d b y a m o l e c u l a r p h y l o g e n y , Vavraia culicis a parasite o f a n o p h e l i n e a n d culicine m o s q u i t o e s w a s found to b e c l o s e s t to T. hominis, raising the possibi
lity o f microsporidian myositis b e i n g a v e c t o r - b o r n e disease ( C h e n e y et al., 2 0 0 0 ) . All the Trachipleistophora infections so far d e t e c t e d have b e e n at an a d v a n c e d stage, only investigated b e c a u s e o f the pain and w e a k n e s s o f m u s c l e s suffered b y the patients. Buskila &
G l a d m a n ( 1 9 9 0 ) reported that 35 % o f AIDS patients
suffer s o m e degree o f myositis. In this p a p e r w e report o n an a p p r o a c h to serological d e t e c t i o n o f T. hominis a n d differentiation o f p o l y s p o r o u s microsporidia b y e n z y m e linked immunosorbent assay (ELISA) and W e s tern blotting. T h e s e tests might b e d e v e l o p e d further to detect early infections o f T. hominis in m a n and thus evaluate its role as a cause o f myositis in AIDS patients.
M A T E R I A L S A N D M E T H O D S
ANTIGEN
M
icrosporidia, s p o r e s o f w h i c h w e r e tested for cross reactivity with T. hominis in ELISAs, W e s t e r n blots or both, are listed with their hosts in T a b l e I. T h o s e o f h u m a n origin w e r e p r o p a gated in vitro in the authors' laboratory after previous isolation from h u m a n tissue. Vavraia culicis w a s prop a g a t e d in large l e p i d o p t e r a n larvae a n d purified spores w e r e provided b y courtesy o f Dr J . J . B e c n e l . T h e other microsporidia, o f fish, c r u s t a c e a n or insect origin, w e r e o b t a i n e d from their natural hosts, either as purified spores or infected host tissues. S p o r e s w e r e purified from culture supernatants or b y m e c h a n i c a l disruption o f host tissue, then lysis o f host cells in water for o n e hour. T h e lysate w a s s p u n at 5 0 0 g for 10 min, the pellet w a s r e s u s p e n d e d in Minimal Essen
tial Medium (MEM) a n d spun o n a 5 0 % Percoll gra
dient in P B S at 9 0 0 g for 3 0 min. After w a s h i n g three
Microsporidia Hosts ELISA1 titre Western blot2
Trachipleistophora hominis Homo sapiens (man)3 12,800 +
Vavraia culicis Aedes alhopictus (mosquito)1 800/1,600 +
Heterosporis anguillarum Anguilla japonica (eel) 200 +
Pleistophora h ippoglossoideos Hippoglossoides platessoides (dab) 400 ND
Pleistophora mirandellae Rutilus rutilus (roach) 800 + / -
Pleistophora typicalis Myoxocephalus scorpius (bull rout) 400 +
Pleistophora sp. (TB) Taurulus bubalis (sea scorpion) 200 +
Pleistophora sp. (LS) Litopenaeus setiferus (shrimp) 800
Pleistophora sp. (FA) Farfantepenaeus aztecus (shrimp) 400 ND
-
Spraguea lophii Lophius americanus (angler fish) 200
Glugea anomala Gasterosteus aculeatus (stickleback) ND
-
-
Lorna acerinae Gymnocephalus cemuus (rufO ND
Encephalitozoon cuniculi Canis familiaris (dog)5 800
-
Encephalitozoon hellem Homo sapiens (man)6 400 ND
-
Vairimorpha mesnili Pieris brassicae (moth) 200 ND
Vittaforma corneae Homo sapiens (man)7 400 ND
ELISA titres are those obtained with mouse serum 483 (titre = 1:12,800).
2Western blotting was carried out with serum 443 (titre - 1:3,200). Results are given as positive (+), negative ( - ) or borderline ( + / - ) . ND = not done.
'Propagated in RK13 cells after isolation from human muscle (Hollister et al, 1 9 9 6 ) . 'Propagated in larvae of Helicoverpa zea (Lepidoptera).
5Propagated in MDCK cells after isolation from dog (Stewart et al., 1979).
6Wainwright isolate, propagated in MDCK cells from human nasal polyp (Hollister et al, 1993).
"Propagated in MDCK cells from human corneal biospy (Shadduck et al, 1990; Silveira & Canning, 1 9 9 5 ) . Table I. - Microsporidia and their hosts used in ELISA and Western blotting.
times the spore pellet was resuspended in MEM and stored at 4 ° C .
ANTISERA
B a l b C euthymic m i c e w e r e inoculated intraperito- neally (ip) or s u b c u t a n e o u s l y ( s c ) with 5 x 1 06 or 107 w h o l e or disrupted s p o r e s o f T. hominis with o r without the adjuvant m o n o p h o s p h o r y l lipid A plus tre
halose dicorynomycolate emulsion (MPL+TDM, Sigma).
T w o to four inoculations w e r e performed according to r e s p o n s e s o f individual mice. Titres w e r e determined by ELISA using the dilution which gave an optical den
sity reading equivalent to twice that o f the control (normal serum) at the starting dilution. A single sample o f human serum from an AIDS patient with T. hominis infection was also tested, with normal human serum as control.
E L I S A
Spores o f e a c h species w e r e added at 2 x 1 05 spores per well (4 x 1 05 spores o f Encephalitozoon cuniculi, w h i c h has smaller s p o r e s ) in c a r b o n a t e / b i c a r b o n a t e buffer overnight at 4 ° C . Non-adherent spores w e r e r e m o v e d with the buffer and the remainder fixed to the plate with a 1:1 mixture o f ethanol and methanol.
T h e b l o c k i n g agent was 1 % horse serum ( H S ) in P B S . Normal m o u s e serum ( c o n t r o l ) and m o u s e antiserum to T. hominis w e r e serially diluted in 1 % HS in P B S from 1/200 to 1 / 1 0 0 , 0 0 0 and incubated with antigen for o n e hour at r o o m temperature ( R T ) . For visualiza
tion V e c t a s t a i n8 ABC-AP kits for m o u s e IgG (Vector Laboratories) and, in o n e experiment the kit for m o u s e IgM, w e r e used according to the manufacturer's ins
tructions and the optical densities w e r e read 10 min and 3 0 min after addition o f substrate in a Dynatek MR 6 0 Microplate reader. Controls w e r e E. coli (strain J M 1 0 9 ) as n o n - m i c r o s p o r i d i a n antigen and coating buffer only in p l a c e o f serum. V e c t a s t a i n1 ABC-AP kits for h u m a n IgG and, in o n e experiment, the kits for human IgM, IgA, IgE and kappa and lambda w e r e used for visualising reactions o f the h u m a n serum.
SDS-PAGE
1 08 s p o r e s o f e a c h s p e c i e s ( 2 x 1 08 spores o f E. cuni
culi) w e r e s u s p e n d e d in 2 0 0 pi o f modified Laemmli reducing buffer ( 5 0 mM TRIS-HC1 pH 6.8, 100 mM dithriothreitol, 10 % glycerol). S p o r e s w e r e disrupted in a b e a d b e a t e r ( B i o s p e c Products), 10 % SDS was added and the sample w a s b o i l e d for 5 min, spun at 1,200 g for 10 min and the supernatant containing the soluble s p o r e proteins was collected. A 10 pi aliquot w a s used for determination o f protein concentration (Protein Assay, B i o R a d ) . B r o m o p h e n o l blue (0.1 % ) was added to the remainder for SDS-PAGE in a BioRad
mini-gel apparatus, using 4 % polyacrylamide stacking gel and 10 % reducing polyacrylamide separating gel.
Samples w e r e added as 10 pi aliquots containing 0.5 pg protein to the slots, as well as 5 pi o f a prestained 10- 2 5 0 kDa molecular weight protein standard (Amer- sham, R a i n b o w Catalogue). Gels w e r e run in at 120 V for 5 min and at 7 0 V for a further 31'2 hours. Gels w e r e silver stained ( B i o R a d ) .
WESTERN BLOTTING
Unstained proteins separated by SDS-PAGE w e r e trans
ferred electrophoretically to Blot PVDF paper, pore size 0.2 p m ( B i o R a d ) in a Mini Trans Blot system ( B i o R a d ) , for 1 h at 100 V at 5° C in running buffer ( 4 8 mM TRIS pH 9.2, 3 9 mM glycine, 2 0 % m e t h a n o l ) . T h e paper with transferred proteins was dipped into methanol, incubated for 1 h in 5 % n o n fat dry milk p o w d e r in PBS, w a s h e d in P B S and incubated overnight with m o u s e or h u m a n antiserum. Reactive proteins w e r e detected with Vectastain® ABC-AF kit (Vector Labora
tories).
R E S U L T S
TlTRES OF MOUSE SERA
U
sing different combinations o f inoculation route (ip or s c ) , w h o l e or dismpted spores o f T. hominis, with or without MPL + TDM, it w a s deter
m i n e d that w h o l e spores inoculated i.p. stimulated a g o o d antibody r e s p o n s e , if given with adjuvant. T h e titres and details o f inoculation are given in T a b l e II.
Serum from m o u s e 4 8 5 was virtually non-reactive and was discarded.
E L I S A
Antiserum 4 8 3 with a titre o f 1:12,800, w a s used to test cross reactivity with the full range o f microsporidia available. T h e titres are given in T a b l e I and s h o w that the serum was strongly reactive o n T. hominis and had s o m e cross reactivity with several o f the other species tested. It was at least eight times more reactive for IgG with T. hominis antigen ( 1 : 1 2 , 8 0 0 ) than with V. culicis (1:800-1,600). Reactivity was less on Pleistophora miran- dellae, Pleistophora sp. (LS) from Litopenaeus setiferus and E. cuniculi ( 1 : 8 0 0 ) , Pleistophora hippoglossoideos, Pleistophora typicalis, Pleistophora sp. ( F A ) from Far- fantepenaeus aztecus, Encephalitozoon hellem and Vit- taforma corneae ( 1 : 4 0 0 ) and was non-reactive o n Hete- rosporis anguillarum, Pleistophora sp. ( T B ) from Taurulus bubalis, Spraguea lophii and Vairimorpha mesnili. W h e n tested for IgM o n T. hominis, the titre o f serum 4 8 3 was 1:800. Serum from the AIDS patient w h o had b e e n infected with T. hominis gave a titre o f
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Mémoire 93
SEROLIGICAL DIFFERENTIATION OF TRACHIPLESTOPHORA
1:800 for I g G in the h o m o l o g o u s test but w a s n e g a tive for IgM, IgA, IgE, k a p p a and l a m b d a light chains.
As there had b e e n s o m e cross reactivity b e t w e e n serum 483 and most o f the microsporidia tested, all the other sera that had b e e n raised in m i c e against T. hominis e x c e p t s e r u m 4 8 5 ( T a b l e I I ) w e r e r e t e s t e d using T. hominis and V. culicis as antigen, to assess cross reactivity with the latter. In this test, serum 4 8 3 w a s eight times m o r e specific for T. hominis ( 1 : 1 2 , 8 0 0 versus 1:1,600) but s e r u m 4 4 3 , with a l o w e r titre against T. hominis, s h o w e d the highest specificity i.e.
I 6 x m o r e reactive o n T. hominis than o n V. culicis ( 1 : 3 , 2 0 0 versus 1:200). T h e highest titre serum ( 4 8 6 ) h a d almost the lowest specificity for T. hominis b e i n g only t w i c e as reactive o n T. hominis as o n V. culicis ( 1 : 2 5 , 8 0 0 versus 1 2 , 8 0 0 ) . Serum 4 4 3 w a s s e l e c t e d for W e s t e r n blotting b e c a u s e o f its high specificity and b e c a u s e it r e v e a l e d a greater n u m b e r o f b a n d s than did other sera in preliminary W e s t e r n blots (data not s h o w n ) .
SDS-PAGE AND WESTERN BLOTS
T h e protein profiles o f 11 microsporidia s e p a r a t e d b y SDS PAGE are s h o w n in Figure 1. T h e b a n d s r e v e a l e d in T. hominis are a g r o u p o f four o r five b e t w e e n 100 and 7 0 kDa, o n e at 6 2 k D a , a g r o u p o f t h r e e b e t w e e n 54 a n d 5 0 kDa, a major b a n d at 4 4 k D a , others bet
w e e n 4 0 and 3 4 k D a , a n o t h e r major b a n d at 32 k D a a n d several b e l o w 3 0 k D a . A clear profile w a s not o b t a i n e d for Pleistophora sp. ( T B ) from T. bubalis. All profiles w e r e unique.
W h e n identical p r o t e i n profiles w e r e b l o t t e d with m o u s e serum 4 4 3 (Fig. 2 ) , major i m m u n o g e n i c anti
g e n s o f T. hominis w e r e displayed b e t w e e n 1 5 0 a n d 9 8 k D a and b e t w e e n 4 4 and 3 9 k D a a n d there w e r e others a b o v e 2 5 0 , at 8 0 , 6 6 , 3 8 , 3 6 , 3 3 a n d b e l o w 25 kDa. Cross reactive antigens w e r e also demonstrated in V. culicis, P. typicalis, Pleistophora sp. ( T B ) from T, bubalis and H. anguillarum but there w a s n o bin
ding to the l o w m o l e c u l a r weight b a n d s o f t h e s e s p e -
Mouse
Number o f s p o r e s per Inoculation
Number o f inoculations
Condition
o f spores Solution Route Titre
443 5 x 1 06 4 whole PBS i.p. 3,200
445 5 x 1 06 4 whole MPL+TDM i.p. 3,200
447 5 x 1 06 4 disrupted MPL+TDM s.c. 6,400
483 1 x 10" 2 whole MPL+TDM i.p. 12,800
484 1 x 10" 3 whole MPL+TDM i.p. 12,800
485 5 x 106 3 whole MPL+TDM i.p. 200
486 5 x 106 3 whole MPL+TDM i.p. 25,600
Table II. - Methods for raising antisera in mice against T. hominis and titres of the sera.
Fig. 1. - Silver-stained SDS-PAGE separated protein profiles of T. hominis in comparison with profiles of eleven microsporidia of fish, crustacean, insect and mammalian origin.
Lanes are: a. Trachipleistophorus hominis, b. Vavraia culicis, c. Pleistophora typicalis, d. Pleistophora sp. (TB) from Taurulus bubalis, e. Heterosporis anguillarum, f. Pleistophora
mirandellae, g. Pleistophora sp. (LS) from Lito- penaeus setiferus, h. Glugea anomala, i. Loma
acerinae, j . Spraguea lophii, k. Encephalito- zoon cuniculi.
CHENEY S.A., LAFRANCHI-TRISTEM N.J. & CANNING E.U.
Fig. 2. - Western blot of SDS PAGE-separated protein profiles of T. hominis and eleven other microsporidia with mouse antiserum 443 raised against T. hominis. Lanes a-k are as in Figure 1.
cies. In V. culicis bands w e r e visible a b o v e 2 5 0 kDa, at 1 7 0 - 1 3 0 kDA and at 8 8 kDa. Major antigens o f 1 4 0 - 100 k D a , 9 0 k D a and 6 5 k D a w e r e identical in P. typi- calis and Pleistophora sp. ( T B ) from T. bubalis. Reac
tive antigens from H. anguillarum w e r e in the region o f 2 5 0 - 1 7 0 k D a and b e t w e e n 130 and 6 5 kDa. A single b a n d at about 135 k D a was given by P. mirandellae.
No antigens in c o m m o n with T. hominis w e r e revealed by antiserum 4 4 3 in Pleistophora sp. (LS) from L. seti- ferus, Glugea anomala, Loma acerinae, S. lophii or
E. cuniculi. Serum from the AIDS patient was non-reac
tive o n T. hominis antigens e v e n at 1:10 dilution.
D I S C U S S I O N
I
n the present study all the microsporidia investigated w e r e found to have u n i q u e protein profiles.
S o m e e p i t o p e s o f i m m u n o g e n i c a n t i g e n s w e r e c o m m o n to Trachipleistophora, Vavraia, Heterosporis and Pleistophora but n o n e o f these w e r e shared b y the remaining genera, Glugea, Loma, Spraguea and Ence- phalitozoon, as revealed in W e s t e r n blots. T h e r e was also n o reactivity with Pleistophora sp. (LS) from the shrimp L. setiferus, suggesting that this species does not b e l o n g to the g e n u s Pleistophora and should b e assi
g n e d to another genus after morphological re-exami
nation.
C o m p a r i s o n o f the ELISA and Western blot results o b t a i n e d with m o u s e anti-r. hominis sera revealed s o m e anomalies. H. anguillarum, P. typicalis and Pleis
tophora sp. ( T B ) gave strong profiles in W e s t e r n blots but w e r e negative or o f very l o w titre b y ELISA. T h e c l o s e relationship b e t w e e n P. typicalis and Pleisto
phora sp. ( T B ) indicated b y phylogenies b a s e d o n 1 6 S rDNA ( C h e n e y et al, 2 0 0 0 ; Nilsen, 2 0 0 0 ) was confirmed b y their very similar profiles in Western blots (Fig. 2 ) . Unfortunately the SDS PAGE profile o f Pleis
tophora sp. ( T B ) was not sufficiently clear to support this. P. mirandellae g a v e an ELISA titre o f 1:800, greater than that o f P. typicalis ( 1 : 4 0 0 ) yet there was barely a reaction in the W e s t e r n blot, e v e n though the SDS PAGE profile, a duplicate o f that w h i c h w a s blotted, revealed a g o o d range o f proteins. Furthermore Pleistophora sp. (LS) and the Encephalitozoon spp.
w e r e moderately positive b y ELISA but totally nega
tive in the Western blot. S o m e o f the l o w titres in the ELISA could have o c c u r r e d b e c a u s e spores w e r e dis
l o d g e d from the plastic walls o f the ELISA plates during processing. W e c o n c l u d e that the ELISA with s e r u m 4 8 3 simply indicated that there w a s strong h o m o l o g o u s reactivity and very little h e t e r o l o g o u s reactivity, without accurate differentiation o f the other species. T h e Western blot results are considered to b e m o r e reliable b e c a u s e the amount o f protein applied to the gels was adjusted to 0.5 ug for e a c h species, making e a c h profile c o m p a r a b l e and b e c a u s e they support the p h y l o g e n e t i c relationships indicated b y s e q u e n c e s o f the 1 6 S rRNA g e n e ( C h e n e y et al., 2 0 0 0 ; Nilsen, 2 0 0 0 ) and o f the R P B 1 g e n e ( C h e n e y et al., in press). T h e 1 6 S rDNA phylogenies s h o w e d that the s p e c i e s w h i c h w e r e strongly cross reactive in the pre
sent Western blot results are m o r e closely related to o n e another than any o f these are to Pleistophora sp.
(LS) or to the g e n e r a Glugea, Loma and Spraguea.
T h e s e relationships w e r e upheld in a phylogeny b a s e d o n s e q u e n c e s o f the A-G region o f the largest subunit o f RNA p o l y m e r a s e ( R P B 1 ) ( C h e n e y et al., in press).
SEROLOGICAL DIFFERENTITATION OF TRACHIPLEISTOPHORA
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Mémoire 95
C H E N E Y S.A., L A F R A N C H I - T R I S T E M N . J . & C A N N I N G E . U .
M o u s e serum 4 4 3 used for Western blotting w a s the only s e a i m that h a d b e e n obtained after i.p. inocula
tion o f spores in P B S . In other c a s e s spores had b e e n administered in adjuvant. Intraperitoneal inoculation has b e e n d e m o n s t r a t e d as a route o f infection in athymic m i c e (Hollister et al., 1 9 9 6 ) . It is likely that in m o u s e 4 4 3 there had b e e n an active, though transient, infection and the serum would h a v e c o n t a i n e d anti
b o d i e s specific for internal s p o r e antigens as well as antigens o f the spore coat. This is supported b y t h e greater n u m b e r o f bands revealed in Western blots b y serum 4 4 3 than b y t h e other sera. Also, as m o u s e serum 4 4 3 w a s used for Western blotting o f proteins from the range o f microsporidian species, while serum 4 8 3 w a s used in the ELISA tests against these species, the results are n o t strictly c o m p a r a b l e . In spite o f the differences, it is clear that T. hominis c a n b e differen
tiated from closely related microsporidia b y its unique SDS-PAGE profile a n d that highly specific antisera raised to T. hominis c o u l d b e used to s c r e e n muscle biopsies from AIDS patients with myositis to detect early infections a n d d e t e r m i n e w h e t h e r or not T.
hominis is involved in the pathology. Such sera w o u l d also differentiate T. hominis from the other m i c r o s p o ridia w h i c h have c a u s e d myositis in AIDS patients i.e.
Pleistophora sp. (Ledford et al., 1 9 8 5 ) and Brachiola vesicularum (Cali et al., 1 9 9 8 ) .
Serum from o n e patient, with severe myositis due to T. hominis, w h o w a s in t h e terminal stages o f AIDS, w a s mildly reactive on T. hominis antigen b y ELISA but non-reactive o n Western blots. In a previous study o f an AIDS patient with E. hellem, from w h o m serum samples had b e e n kept over a period o f four years, there had b e e n a marked drop in antibody level in the terminal stages o f AIDS, as revealed b y W e s t e r n blot
ting o f culture-derived spore antigens (Hollister et al., 1 9 9 3 ) : most o f the characteristic proteins o f this spe
cies s h o w n b y the serum samples taken earlier in the patient's illness w e r e n o longer r e c o g n i s e d b y the last sample. In the description o f E. hellem w h i c h w a s dif
ferentiated from E. cuniculi b y SDS-PAGE and W e s tern blotting (Didier et al., 1 9 9 1 ) , sera from three patients with keratoconjunctivitis due to E. hellem w e r e tested o n SDS-PAGE protein profiles o f in vitro cul
ture-derived s p o r e s o f E. hellem. Serum from o n e patient w h o was at a slightly earlier stage o f AIDS reacted strongly against t h e E. hellem protein profiles, w h e r e a s the reactions o f the sera o f the other t w o patients, w h o w e r e in the terminal stages o f AIDS, w e r e much w e a k e r . Although these sera w e r e derived from separate individuals, w h o s e i m m u n e r e s p o n s e s would not h a v e b e e n identical, the results are in a c c o r d with the decline in antibody level, w h i c h was tracked b y Hollister et al. ( 1 9 9 3 ) in o n e patient, as AIDS pro
gressed. T h e s e results suggest that late stage sera might not b e useful in diagnosis o f microsporidial infections
such as T. hominis or T. anthropophthera, as these infections are o n l y likely t o b e p r o b l e m a t i c ( a n d d e t e c t e d ) in the terminal stages o f AIDS. However, should T. hominis b e d e t e c t e d in AIDS patients in future, retrospective examination o f sera obtained at an earlier stage o f AIDS might reveal high levels o f spe
cific antibodies. Such sera w o u l d b e valuable in detec
tion o f T. hominis antigen in b i o p s i e s or as a control in serological surveys o f patients with myositis, using the patient's sera against culture derived T. hominis antigen.
ACKNOWLEDGEMENTS
T
h e authors w o u l d like to thank t h e following p e o p l e for provision o f s p o r e s o f t h e microsporidia u s e d in this study. D r s J . J . B e c n e l (y, culicis), J . V . M a d d o x (V. mesnili), K. M c K e n z i e {P. hippoglossoideos), R.M. Overstreet {Pleistophora sp.
LS and FA), M. Pekkarinen {P. mirandellae a n d L. ace- rinae), E. Weidner ( 5 . lophii), H. Y o k o y a m a (H. anguil- larum) a n d R. Turner (P. typicalis). W e are also gra
teful to D r S.W. Feist a n d M. L o n g s h a w for assistance in collection o f Taurulus bubalis and D r B . Currie for provision o f serum from the patient with the T. hominis infection. T h e work was funded b y the Medical Research Council, UK (Grant No. G 9 6 0 3 0 5 0 P B ) .
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Reçu le 16 septembre 2000 Accepté le 27 novembre 2000
Mémoire
SEROLOGICAL DIFFERENTIATION OF TRACHIPLEISTOPHORA
9 7
Parasite, 2001, 8, 91-97
