Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2001082091
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 . *
Summary: 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.
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 3944 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 admission to hospital o f progressive HIV
INTRODUCTION
disease without
T
he
microsporidian
hominis
Hollister,
parasite
Trachipleistophora
Canning,
Weidner,
Field,
K e n c h & Marriott, 1 9 9 6 w a s found infecting the
skeletal m u s c l e a n d c o r n e a and w a s present in nasal discharge o f an AIDS patient (Field et al., 1 9 9 6 ) . T h e patient had 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 muscular pain and wasting but his condition improved after treatm e n t with a therapeutic c o m b i n a t i o n w h i c h included 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 anti-microsporidial
activity. T h e
patient
died
five
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 and d e s c r i b e d as a n e w g e n u s and s p e c i e s (Hollister et al.,
1996).
In n u m e r o u s g e n e r a o f microsporidia, s p o r o g o n y culminates
in a large ( m o r e than e i g h t ) and
s u m e d parasite origin ( p o l y s p o r o u s in
and Glugea,
Heterosporis
and Pleistophora
k n o w n from vertebrates Pleistophora-like also infect e d i b l e crustaceans and Heterosporis
from m a n from fish are microsporidia
shrimps
and
hypertrophied
host cells modified to form x e n o m a s . Pleistophora Trachipleistophora g e n e r a Loma In Loma,
crabs.
infections are strictly localised
in their hosts b y b e i n g retained within
Parasite, 2001, 8, 91-97
sporophorous
v e s i c l e s ) . O f t h e s e o n l y Trachipleistophora
Glugea
* 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]
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-
and
infections are diffuse. Species o f the
and Spraguea
are also parasites o f fish.
s p o r o g o n y gives rise to four or m o r e spores
retained within a parasitophorous v a c u o l e b o u n d e d b y
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suffer s o m e degree o f myositis. In this paper w e report o n an approach to serological detection o f T. hominis and differentiation o f polysporous microsporidia b y enzyme linked immunosorbent assay (ELISA) and Wes 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 man and thus evaluate its role as a cause o f myositis in AIDS patients.
a m e m b r a n e o f presumed host origin (Lom & Pekkarinen, 1 9 9 9 ) . In Spraguea there are two sporogonic s e q u e n c e s which give rise to t w o types o f spores, both lying free in host cell cytoplasm ( n o enveloping mem brane). 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 myositis in AIDS patients, the parasites w e r e identified as Pleistophora spp. (Chupp et al, 1993; Grau et al, 1 9 9 6 ) but are more 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 diagnosed recently (A. Curry, personal c o m m u n i c a t i o n ) . A s e c o n d species Trachipleistophora anthropophthera has c a u s e d a multisystem dissemina ting infection, especially infecting the brain, in two m o r e AIDS patients ( Y a c h n i s et al., 1996; Vavra et al, 1998). T h e o c c u r r e n c e o f several o f these d e e p tissue infections, w h i c h cannot b e transmitted directly bet w e e n humans, at least not from muscle 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 revealed b y a molecular phylogeny, Vavraia culicis a parasite o f a n o p h e l i n e and culicine mosquitoes w a s found to b e closest to T. hominis, raising the possibi lity o f microsporidian myositis being a vector-borne disease (Cheney et al., 2000). All the Trachipleistophora infections so far detected have b e e n at an advanced stage, only investigated b e c a u s e o f the pain and weak ness o f muscles suffered b y the patients. Buskila & Gladman ( 1 9 9 0 ) reported that 35 % o f AIDS patients
Microsporidia Trachipleistophora hominis Vavraia culicis Heterosporis anguillarum Pleistophora h ippoglossoideos Pleistophora mirandellae Pleistophora typicalis Pleistophora sp. (TB) Pleistophora sp. (LS) Pleistophora sp. (FA) Spraguea lophii Glugea anomala Lorna acerinae Encephalitozoon cuniculi Encephalitozoon hellem Vairimorpha mesnili Vittaforma corneae
MATERIALS A N D M E T H O D S ANTIGEN
M
icrosporidia, spores o f w h i c h w e r e tested for cross reactivity with T. hominis in ELISAs, Western 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 propa
gated in vitro in the authors' laboratory after previous isolation from h u m a n tissue. Vavraia culicis w a s pro p 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, crustacean or insect origin, w e r e obtained from their natural hosts, either as purified spores or infected host tissues. Spores 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 spun at 5 0 0 g for 10 min, the pellet w a s resuspended in Minimal Essen tial Medium (MEM) and spun on a 50 % Percoll gra dient in P B S at 9 0 0 g for 3 0 min. After washing three
1
Hosts 3
Homo sapiens (man) Aedes alhopictus (mosquito) Anguilla japonica (eel) Hippoglossoides platessoides (dab) Rutilus rutilus (roach) Myoxocephalus scorpius (bull rout) Taurulus bubalis (sea scorpion) Litopenaeus setiferus (shrimp) Farfantepenaeus aztecus (shrimp) Lophius americanus (angler fish) Gasterosteus aculeatus (stickleback) Gymnocephalus cemuus (rufO Canis familiaris (dog) Homo sapiens (man) Pieris brassicae (moth) Homo sapiens (man)7 1
5
6
ELISA titre
Western blot
12,800 800/1,600 200 400 800 400 200 800 400 200 ND ND 800 400 200 400
+ + + ND +/+
2
+
ND
ND
ND ND
ELISA titres are those obtained with mouse serum 483 (titre = 1:12,800). Western 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). Propagated in MDCK cells after isolation from dog (Stewart et al., 1979). Wainwright 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 ) . 2
5
6
Table I. - Microsporidia and their hosts used in ELISA and Western blotting.
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SEROLIGICAL DIFFERENTIATION OF TRACHIPLESTOPHORA
times the spore pellet was resuspended in MEM and stored at 4 ° C . ANTISERA Balb C euthymic mice w e r e inoculated intraperitoneally (ip) or subcutaneously ( s c ) with 5 x 1 0 or 107 w h o l e or disrupted s p o r e s o f T. hominis with or without the adjuvant monophosphoryl lipid A plus tre halose dicorynomycolate emulsion (MPL+TDM, Sigma). T w o to four inoculations were performed according to responses o f individual mice. Titres were 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 of human serum from an AIDS patient with T. hominis infection was also tested, with normal human serum as control. 6
ELISA 5
Spores o f e a c h species w e r e added at 2 x 1 0 spores per well (4 x 1 0 spores o f Encephalitozoon cuniculi, which has smaller spores) in carbonate/bicarbonate buffer overnight at 4°C. Non-adherent spores w e r e removed with the buffer and the remainder fixed to the plate with a 1:1 mixture o f ethanol and methanol. T h e blocking agent was 1 % horse serum ( H S ) in PBS. Normal m o u s e serum (control) 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 1/100,000 and incubated with antigen for o n e hour at room temperature (RT). For visualiza tion V e c t a s t a i n ABC-AP kits for m o u s e IgG (Vector Laboratories) and, in o n e experiment the kit for mouse IgM, were used according to the manufacturer's ins tructions and the optical densities were 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 non-microsporidian antigen and coating buffer only in place o f serum. V e c t a s t a i n ABC-AP kits for human IgG and, in o n e experiment, the kits for human IgM, IgA, IgE and kappa and lambda were used for visualising reactions o f the human serum.
mini-gel apparatus, using 4 % polyacrylamide stacking gel and 10 % reducing polyacrylamide separating gel. Samples were added as 10 pi aliquots containing 0.5 pg protein to the slots, as well as 5 pi o f a prestained 10250 kDa molecular weight protein standard (Amersham, Rainbow Catalogue). Gels were run in at 120 V for 5 min and at 7 0 V for a further 3 ' hours. Gels w e r e silver stained (BioRad). 1
2
WESTERN BLOTTING Unstained proteins separated by SDS-PAGE were trans ferred electrophoretically to Blot PVDF paper, pore size 0.2 pm (BioRad) in a Mini Trans Blot system (BioRad), for 1 h at 100 V at 5° C in running buffer ( 4 8 mM TRIS pH 9.2, 3 9 mM glycine, 20 % methanol). T h e paper with transferred proteins was dipped into methanol, incubated for 1 h in 5 % non fat dry milk powder in PBS, washed in P B S and incubated overnight with m o u s e or human antiserum. Reactive proteins were detected with Vectastain® ABC-AF kit (Vector Labora tories).
5
8
1
SDS-PAGE 8
8
1 0 spores o f e a c h species (2 x 1 0 spores o f E. cuni culi) w e r e suspended in 200 pi o f modified Laemmli reducing buffer ( 5 0 mM TRIS-HC1 pH 6.8, 100 mM dithriothreitol, 10 % glycerol). Spores w e r e disrupted in a b e a d beater ( B i o s p e c Products), 10 % SDS was added and the sample was boiled for 5 min, spun at 1,200 g for 10 min and the supernatant containing the soluble spore proteins was collected. A 10 pi aliquot was used for determination o f protein concentration (Protein Assay, BioRad). 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 Parasite, 2001, 8, 91-97
RESULTS TlTRES OF MOUSE SERA
U
sing different combinations o f inoculation route (ip or sc), whole or dismpted spores o f T. homi nis, with or without MPL + TDM, it was deter mined that w h o l e spores inoculated i.p. stimulated a g o o d antibody response, if given with adjuvant. T h e titres and details o f inoculation are given in Table II. Serum from m o u s e 4 8 5 was virtually non-reactive and was discarded. ELISA
Antiserum 4 8 3 with a titre o f 1:12,800, was used to test cross reactivity with the full range o f microsporidia available. T h e titres are given in Table I and s h o w that the serum was strongly reactive on 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 mirandellae, Pleistophora sp. (LS) from Litopenaeus setiferus and E. cuniculi ( 1 : 8 0 0 ) , Pleistophora hippoglossoideos, Pleistophora typicalis, Pleistophora sp. (FA) from Farfantepenaeus aztecus, Encephalitozoon hellem and Vittaforma corneae (1:400) and was non-reactive on Heterosporis anguillarum, Pleistophora sp. ( T B ) from Taurulus bubalis, Spraguea lophii and Vairimorpha mesnili. W h e n tested for IgM on 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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1:800 for IgG in the h o m o l o g o u s test but w a s nega tive for IgM, IgA, IgE, kappa and lambda 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 was eight times m o r e specific for T. hominis (1:12,800 versus 1:1,600) but serum 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 on V. culicis (1:3,200 versus 1:200). T h e highest titre serum ( 4 8 6 ) had almost the lowest specificity for T. hominis being only twice as reactive on T. hominis as on V. culicis ( 1 : 2 5 , 8 0 0 versus 1 2 , 8 0 0 ) . Serum 4 4 3 w a s selected for Western blotting b e c a u s e o f its high specificity and b e c a u s e it revealed a greater n u m b e r o f bands than did other sera in preliminary Western blots (data not shown).
Mouse 443 445 447 483 484 485 486
Number of spores per Inoculation 5 5 5 1 1 5 5
x 10 x 10 x 10 x 10" x 10" x 10 x 10 6
6
6
6
6
SDS-PAGE AND WESTERN BLOTS T h e protein profiles o f 11 microsporidia separated b y SDS PAGE are s h o w n in Figure 1. T h e bands revealed in T. hominis are a group o f four or five b e t w e e n 100 and 7 0 kDa, o n e at 6 2 kDa, a group o f three b e t w e e n 54 and 50 kDa, a major b a n d at 4 4 kDa, others bet w e e n 4 0 and 34 kDa, another major b a n d at 32 kDa and several b e l o w 30 kDa. A clear profile was not obtained for Pleistophora sp. ( T B ) from T. bubalis. All profiles w e r e unique. W h e n identical protein 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 gens o f T. hominis w e r e displayed b e t w e e n 1 5 0 and 98 kDa and b e t w e e n 4 4 and 3 9 k D a and there w e r e others a b o v e 2 5 0 , at 8 0 , 6 6 , 3 8 , 3 6 , 33 and b e l o w 25 kDa. Cross reactive antigens were 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 molecular weight bands o f these spe-
Number of inoculations
Condition of spores
Solution
Route
Titre
4 4 4 2 3 3 3
whole whole disrupted whole whole whole whole
PBS MPL+TDM MPL+TDM MPL+TDM MPL+TDM MPL+TDM MPL+TDM
i.p. i.p. s.c. i.p. i.p. i.p. i.p.
3,200 3,200 6,400 12,800 12,800 200 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 Litopenaeus setiferus, h. Glugea anomala, i. Loma acerinae, j . Spraguea lophii, k. Encephalitozoon cuniculi.
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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 were visible a b o v e 2 5 0 kDa, at 170-130 kDA and at 8 8 kDa. Major antigens o f 140100 kDa, 90 kDa and 65 kDa w e r e identical in P. typicalis and Pleistophora sp. ( T B ) from T. bubalis. Reac tive antigens from H. anguillarum were in the region o f 250-170 kDa and b e t w e e n 130 and 65 kDa. A single band at about 135 kDa was given by P. mirandellae. No antigens in c o m m o n with T. hominis were revealed by antiserum 4 4 3 in Pleistophora sp. (LS) from L. setiferus, Glugea anomala, Loma acerinae, S. lophii or E. cuniculi. Serum from the AIDS patient was non-reac tive o n T. hominis antigens even at 1:10 dilution.
DISCUSSION
I
n the present study all the microsporidia investi gated were found to have unique 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 antigens 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 by the remaining genera, Glugea, Loma, Spraguea and Encephalitozoon, as revealed in Western blots. There 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 genus Pleistophora and should b e assi g n e d to another genus after morphological re-exami nation. Comparison o f the ELISA and Western blot results obtained 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 Western blots but w e r e negative or o f very l o w titre by ELISA. T h e close relationship b e t w e e n P. typicalis and Pleisto Parasite, 2001, 8, 91-97
Mémoire
phora sp. ( T B ) indicated by phylogenies b a s e d on 1 6 S rDNA ( C h e n e y et al, 2000; Nilsen, 2 0 0 0 ) was confirmed by 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 gave an ELISA titre o f 1:800, greater than that o f P. typicalis (1:400) yet there was barely a reaction in the Western blot, even though the SDS PAGE profile, a duplicate o f that w h i c h was blotted, revealed a good range of proteins. Furthermore Pleistophora sp. (LS) and the Encephalitozoon spp. were moderately positive by ELISA but totally nega tive in the Western blot. S o m e o f the low titres in the ELISA could have occurred b e c a u s e spores w e r e dis lodged from the plastic walls o f the ELISA plates during processing. W e conclude that the ELISA with serum 4 8 3 simply indicated that there was 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 each species, making each profile comparable and b e c a u s e they support the phylogenetic relationships indicated by s e q u e n c e s o f the 1 6 S rRNA g e n e (Cheney et al., 2000; 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 showed that the species which were strongly cross reactive in the pre sent Western blot results are more closely related to o n e another than any o f these are to Pleistophora sp. (LS) or to the genera Glugea, Loma and Spraguea. T h e s e relationships were upheld in a phylogeny based on s e q u e n c e s o f the A-G region o f the largest subunit o f RNA polymerase ( R P B 1 ) ( C h e n e y et al., in press).
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Mouse serum 4 4 3 used for Western blotting w a s the only s e a i m that had b e e n obtained after i.p. inocula tion o f spores in P B S . In other cases spores had b e e n administered in adjuvant. Intraperitoneal inoculation has b e e n demonstrated as a route o f infection in athymic mice (Hollister et al., 1996). 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 have contained anti bodies specific for internal spore antigens as well as antigens o f the spore coat. This is supported b y the greater n u m b e r o f bands revealed in Western blots b y serum 4 4 3 than b y the 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 483 w a s used in the ELISA tests against these species, the results are not strictly comparable. In spite o f the differences, it is clear that T. hominis can b e differen tiated from closely related microsporidia by its unique SDS-PAGE profile and that highly specific antisera raised to T. hominis could b e used to screen muscle biopsies from AIDS patients with myositis to detect early infections and determine w h e t h e r or not T. hominis is involved in the pathology. Such sera would also differentiate T. hominis from the other microspo ridia which have caused myositis in AIDS patients i.e. Pleistophora sp. (Ledford et al., 1 9 8 5 ) and Brachiola vesicularum (Cali et al., 1998). Serum from o n e patient, with severe myositis due to T. hominis, w h o w a s in the terminal stages o f AIDS, was mildly reactive on T. hominis antigen b y ELISA but non-reactive o n Western blots. In a previous study of 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 by Western blot ting o f culture-derived spore antigens (Hollister et al., 1993): 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 recognised by the last sample. In the description o f E. hellem which w a s dif ferentiated from E. cuniculi by 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 were tested o n SDS-PAGE protein profiles o f in vitro cul ture-derived spores 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 the E. hellem protein profiles, whereas the reactions o f the sera o f the other two patients, w h o were in the terminal stages o f AIDS, were much weaker. Although these sera w e r e derived from separate individuals, w h o s e immune responses would not have b e e n identical, the results are in accord with the decline in antibody level, which was tracked by 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
96
such as T. hominis or T. anthropophthera, as these infections are only likely to b e p r o b l e m a t i c ( a n d detected) in the terminal stages o f AIDS. However, should T. hominis b e detected 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 would b e valuable in detec tion o f T. hominis antigen in biopsies 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
he authors would like to thank the following p e o p l e for provision o f spores o f the micro sporidia u s e d in this study. Drs J . J . B e c n e l (y, culicis), J . V . M a d d o x (V. mesnili), K. McKenzie {P. hippoglossoideos), R.M. Overstreet {Pleistophora sp. LS and FA), M. Pekkarinen {P. mirandellae and L. acerinae), E. Weidner (5. lophii), H. Y o k o y a m a (H. anguillarum) and R. Turner (P. typicalis). W e are also gra teful to Dr S.W. Feist and M. Longshaw for assistance in collection o f Taurulus bubalis and Dr B . Currie for provision o f serum from the patient with the T. hominis infection. T h e work was funded by 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 Parasite, 2001, 8, 91-97
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