IN VITRO PRODUCTION
AND CHARACTERIZATION OF EXCRETORY/SECRETORY PRODUCTS OF ONCHOCERCA VOLVULUS
SAKWE A.M.*, EVEHE M.-S.B.** & TITANJI V . P . K . *,. **
S u m m a r y :
Onchocerca volvulus excretory/secretory products (ESP) free of host contaminants are often needed for immunologic and biochemical studies. Since prolonged in vitro survival of O. volvulus in culture requires the presence of human serum, as a supplement, it was necessary to investigate other culture medium supplements in which pure ESP could be generated. Thus heat- inactivated normal rabbit serum, fetal calf serum and the synthetic serum substitute Ultroser-G were tested for their abilities to sustain O. volvulus adult females and nodular microfilariae in vitro and their abilities to promote the synthesis and release of ESP. Using [35S]-methionine as the radioactive precursor, O. volvulus nodular microfilariae and adult females were shown to actively synthesize and release excretory/secretory proteins with increasing efficiency in human serum, rabbit serum, Ultroser-G and fetal calf serum.
Analysis of the ESP by SDS-PAGE revealed that at least 30 polypeptides in the 7-174 kDa range were continuously released. About 14 of these components were immunogenic to the human host as shown by immunoprecipitation. Prominent antigenic polypeptides were those with relative molecular weights of 13, 16, 3 3 , 6 8 and 170. Rabbit serum, fetal calf serum and Ultroser- G could therefore, conveniently replace human serum in cultures of O. volvulus nodular microfilariae and adult females.
KEY WORDS : Onchocerca volvulus, in vitro culture, excretory/secretory products, Ultroser-G.
MOTS CLÉS : Onchocerca volvulus, culture in vitro, produits d'excrétion/
sécrétion, Ultroser-G.
Résumé : PRODUCTION ET CARACTÉRISATION IN VITRO DES PRODUITS D'EXCRÉTION/SÉCRÉTION D'ONCHOCERCA VOLVULUS
Les produits d'excrétion/sécrétion (PSE) d'Onchocerca volvulus exempts d'impuretés de l'hôte sont utiles pour les études immunologiques et biochimiques. Cependant la survie prolongée d'O. volvulus en culture in vitro nécessite la présence de sérum humain comme additif au milieu de culture. Mais les PSE obtenus avec du sérum humain sont souvent contaminés par des composants du sérum. Il est donc indispensable d'évaluer d'autres additifs de milieu de culture pour lesquels des PSE plus purs peuvent être générés. Ainsi, le sérum normal de lapin inactivé à la chaleur, le sérum de veau fœtal, le sérum humain et l'additif synthétique Ultroser-G ont été testés pour leur capacité à maintenir in vitro les femelles adultes et les microfilaires nodulaires
d'O. volvulus ainsi que pour leur capacité a promouvoir la synthèse et la libération des PSE. En utilisant la [3SS]-méthionine comme précurseur radioactif, les deux stades du parasite étudiés ont montré une synthèse et une libération active de PSE avec une efficacité croissante pour les milieux de culture contenant respectivement comme additif le sérum humain, le sérum de lapin, l'additif synthétique Ultroser-G et le sérum de veau fœtal. L'analyse des PSE par SDS-PAGE a révélé qu'au moins 30 polypeptides de taille allant de 7 à 174 kDa sont continuellement libérés. Par immunoprécipitation, il a été démontré qu'au moins 14 de ces polypeptides sont immunogéniques pour l'homme. Les principaux polypeptides antigéniques ont pour poids moléculaires relatifs :
13, 16, 33, 68 et 170 kDa. Le sérum de lapin, le sérum de veau fœtal et l'Ultroser-G peuvent ainsi convenablement remplacer le sérum humain pour la maintenance in vitro de ces stades d'O.
volvulus et pour l'obtention de PSE moins contaminés.
INTRODUCTION
P
arasitic filariae release into the tissues a n d b o d y fluids o f their hosts e x c r e t o r y - s e c r e t o r y pro- ducts ( E S P ) which circulate freely (Ouaissi et al, 1 9 8 1 ; D e s Moutis et al, 1 9 8 3 ; Petralanda et al, 1 9 8 8 ; Maizels et al, 1 9 9 0 ) or as i m m u n e c o m p l e x e s ( P a g a - nelli et al, 1 9 8 0 ; Steward et al, 1 9 8 2 ) . T h e origin o f t h e s e ESP a p p e a r s to b e diverse, ranging from uterine s e c r e t i o n s o f gravid adult females to surface-associated c o m p o n e n t s r e l e a s e d as a result o f the d y n a m i c nature* Faculty o f Science, University o f Buea, PO B o x 63, Cameroon.
** Biotechnology Centre, University of Yaoundé-1, Cameroon.
Correspondence : Vincent P.K. Titanji*
Parasite, 1997, 4, 351-358
o f t h e cuticle ( H a q u e & Capron, 1 9 8 6 ) . S o m e o f t h e s e ESP h a v e b e e n found to b e b i o l o g i c a l l y active m o l e - c u l e s s u c h as e n z y m e s (Harnett et al, 1 9 8 9 ) w h i c h aid in t h e migration o f t h e s e t i s s u e - d w e l l i n g parasites through host tissues. B y virtue o f their direct c o n t a c t with the host i m m u n e system, they m a y also persis- tently stimulate the host i m m u n e system, causing local inflammatory r e s p o n s e s (Maizels et al, 1 9 8 2 ; Ottessen, 1 9 8 4 ; Philipp étal, 1 9 8 8 ) . Furthermore, ESP act as t h e most reliable indicators o f current infection or the pre- s e n c e o f v i a b l e parasites in the host (Selkirk et al, 1 9 8 6 ) .
О. volvulus has b e e n m a i n t a i n e d in culture for t w o to three w e e k s in m e d i u m s u p p l e m e n t e d with h u m a n s e r u m (Ngu et al, 1 9 8 1 ; E n g e l b r e c h t & Schulz-Key, 1 9 8 4 ; W a l t e r , 1 9 8 8 ) w i t h r e g u l a r c h a n g e s o f t h e
Mémoire - 3 5 1
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1997044351
S A K W E A . M . , E V E H E Μ . - S . B . & T I T A N J I V . P . K .
medium. P r o l o n g e d survival o f the parasite in vitro is h o w e v e r , required for m e t a b o l i c studies as well as for the testing o f anti-filarial products. B a s e d o n the hypo
thesis that the in vitro released products in culture media may c o r r e s p o n d to those synthesised in vivo, culture supernatants have b e e n c o n s i d e r e d as a rich s o u r c e o f ESP (Selkirk et al, 1 9 8 6 ) . T h e frequent approach to the collection and characterization o f ESP is therefore, b y the in vitro culture o f viable parasites for several hours in the presence or not o f radiolabelled precursor a m i n o acids.
Several studies have demonstrated that filarial ESP are immunologically important though fewer in n u m b e r than the corresponding somatic antigens (Maizels et al, 1 9 8 2 ; Ottessen, 1 9 8 4 ; Selkirk et al, 1 9 8 6 ) . However, ESP derived from medium s u p p l e m e n t e d with human serum are not suitable for further work especially for the immunization o f experimental animals b e c a u s e o f the p r e s e n c e o f contaminating human serum proteins.
T h e aim o f this study was to investigate conditions for the in vitro production o f pure ESP.
MATERIALS AND METHODS
PARASITES
N
odular microfilariae (mf) w e r e isolated from fresh nodules by dissection o f the latter under sterile conditions. T h e emergent m f w e r e then purified by centrifugation o n percoli (Pharmacia L K B ) gradients ( C h a n d r a s h e k e r et al., 1 9 8 4 ; Titanji et al., 1 9 8 7 ) . Adult female w o r m s w e r e on the other hand o b t a i n e d from u n d a m a g e d fresh nodules treated with c o l l a g e n a s e from Clostridium histolytica ( B o e r h i n g e r , Mannheinn), under aseptic conditions (Engelbrecht &Schulz-Key, 1 9 8 4 ) . T h e isolated w o r m s w e r e then sub- cultured for 2-3 hours and the viability ascertained by observation o f the m o v e m e n t s o f the adult parasites with the n a k e d e y e and that o f the nodular m f under the m i c r o s c o p e . After isolation, the viability e x c e e d e d 9 8 % for the nodular m f and was 100 % for the adult female worms.
ANTISERA
Human o n c h o c e r c i a s i s patient sera w e r e p r e p a r e d from b l o o d o b t a i n e d from c o n s e n t i n g nodule donors living in a h y p e r - e n d e m i c area in the SA'A sub-Divi
sion in the Sanaga River basin ( C a m e r o o n ) . A pool was made from the sera o f 54 patients as a hyper-immune patient s e n i m pool. Normal human serum pool was s i m i l a r l y r e c o n s t i t u t e d f r o m b l o o d d o n a t e d b y 10 S w e d e s with n o sign o f the disease.
IN VITRO C U L T U R E OF N O D U L A R MF IN T H E P R E S E N C E OF U L T R O S E R - G
Nodular m f ( 1 , 0 0 0 - 2 , 0 0 0 ) in a total v o l u m e o f 1.5 ml.
w e r e cultured in medium RPMI 1 6 4 0 ( G i b c o ) contain
ing 0.16 mg/ml gentamycin and supplemented with the synthetic serum substitute, Ultroser-G at 1, 2 or 4 %.
Medium RPMI 1 6 4 0 containing the antibiotic only o r the s a m e medium s u p p l e m e n t e d with 10 % human serum w e r e used as controls. T h e cultures w e r e main
tained at room temperature under sterile conditions for as long as 7 2 hours during w h i c h the viability o f the parasites was determined as a function o f the incuba
tion time.
METABOLIC LABELLING OF PARASITES
All in vitro culture o f parasites w e r e carried out under sterile conditions. T h e p r o c e d u r e that follows, repre
sents the final protocol adopted for a typical experi
ment. T h e typical e x p e r i m e n t consisted o f performing simultaneously, all the six assays with o n e adult female w o r m per assay, after a series o f trials to determine the optimal concentration o f e a c h supplement, the incubation time as well as the reaction v o l u m e . In this procedure, the parasites w e r e selectively starved from the precursor a m i n o acid, m e t h i o n i n e by main
taining them in methionine-free medium, i.e. EMEM without methionine and glutamine, (Flow laboratories) containing 0 . 1 6 m g / m l gentamycin for 3 hours at r o o m temperature. After washing twice in the s a m e medium, the parasites w e r e then re-suspended in 1.5 ml for 6 , 0 0 0 - 1 0 , 0 0 0 nodular mf or 2.5 ml per adult female o f the labelling medium (methionine-free medium contai
ning 0 . 1 6 m g / m l gentamycin, 4 mM glutamine). T o e a c h assay was added either o f the following medium supplements : 10 % normal human serum (NHS), 10 % heat-inactivated normal rabbit serum (NRS), 10 % fetal calf serum (FCS), o r 1 % Ultroser-G. In a n o t h e r assay, the t w o parasite stages w e r e selectively starved from methionine, pre-incubated at 4 2 °C for 2 0 min (heat- s h o c k t r e a t m e n t ) in the labelling m e d i u m s u p p l e m e n t e d with 10 % FCS.
T h e radioactive precursor, [3 5S]-methionine (Amersham, SJ 1 2 3 ) w a s a d d e d at 2 0 0 - 2 5 0 μα per assay, and labelling performed for 24 hours either at 37 °C or at r o o m temperature ( 2 0 - 2 5 °C). During the labelling, ali- quots o f 100 μΐ w e r e taken from the adult female cul
tures following a time c o u r s e and p r o c e s s e d for liquid scintillation counting ( s e e b e l o w ) . At the e n d o f the labelling, the media from the adult female cultures w e r e transferred to n e w tubes and then centrifuged at 5,000 χ g for 10 min at 4 °C to pellet the in vitro released microfilariae. T h e nodular m f cultures w e r e also centrifuged as above. T h e supernatant in each c a s e constituted the pool o f adult female ESP and nodular
E X C R E T O R Y / S E C R E T O R Y PRODUCTS O F O. VOLVULUS
mf ESP respectively. T o inhibit proteolysis, freshly pre
pared phenymethyl sulfonylfluoride (PMSF) was added to e a c h tube to a final concentration o f 1 Mm before storage in aliquots at - 2 0 °C.
T h e nodular mf pellet and the adult female w o r m s w e r e w a s h e d twice in methionine-free medium and stored frozen at - 2 0 °C until required for the extrac
tion o f antigens. During the entire labelling period, adult females released eggs s o m e o f w h i c h d e v e l o p e d into microfilariae. T h e s e in vitro released eggs and nodular m f w e r e separated and purified over percoli gradients (Titanji et al, 1 9 8 7 ) . Somatic antigens w e r e prepared from the radiolabelled nodular m f and the purified in vitro released eggs as previously described (Titanji et al, 1 9 9 0 ) .
SCINTILLATION COUNTING
T h e aliquots taken from the adult female cultures at various time points w e r e immediately centrifuged at 1 0 , 0 0 0 X g for 5 min at 4 °C to pellet the m f and the supernatant transferred into c l e a n tubes. T h e s e w e r e precipitated with 10 % trichloroacetic acid (TCA) in the p r e s e n c e o f 10 % normal h u m a n serum or normal rabbit serum as carrier proteins. T h e precipitates w e r e w a s h e d thrice with P B S , then air-dried and redissolved in a scintillation cocktail comprising 0.05 % P O P O P , 0.7 % P P O , 3 5 % triton X 1 0 0 in scintillation grade toluene for counting using a Packard ( 2 0 0 2 ) liquid scin
tillation counter.
IMMUNOPRECIPITATION
T h e frozen radiolabelled ESP w e r e t h a w e d and either used directly or cleared with NHS. In the latter option, the immune c o m p l e x e s w e r e removed by precipitation with 10 % w a s h e d pansorbin (Staphylococcus aureus stock. C a l b i o c h e m ) . After centrifugation at 3,000 x g for 15 min at 4 °C, the supernatant constituted the cleared ESP.
Radioimmunoprecipitation was performed as described (Kessler, 1 9 7 5 ) with s o m e modifications (Titanji &
Ngu, 1 9 8 6 ) using the hyper-immune patient serum p o o l and the control normal human serum pool. T h e i m m u n e c o m p l e x e s w e r e p r e c i p i t a t e d with 10 % w a s h e d pansorbin, w a s h e d thrice with i m m u n o p r e c i pitation buffer ( 5 0 mM Tris-HCl pH 7.4, 140 mM NaCl, 5 mM EDTA, 0.02 % sodium azide) and analysed by sodium dodecyl sulphate polyacrylamide gel electro
phoresis (SDS-PAGE).
ELECTROPHORESIS, FLUOROGRAPHY AND AUTORADIOGRAPHY
Electrophoresis o f ESP, somatic extracts from nodular m f and in vitro released eggs and i m m u n e c o m p l e x e s was performed by SDS-PAGE under reducing condi
tions according to the m e t h o d o f Laemmli ( 1 9 7 0 ) in either 6-20 % gradient o r 10 % slab gels. T h e SDS gels w e r e loaded with samples o n the basis o f TCA preci- pitable c m p ( 1 0 - 1 5 x 1 03 c p m w e r e loaded per w e l l ) and Pharmacia l o w molecular weight standards w e r e applied in parallel lanes. After electrophoresis, the gels w e r e fixed for 1 hour in 10 % TCA, stained with C o o m a s s i e brilliant b l u e R-250 and then destained.
Fluorography and drying w e r e performed according to the m e t h o d o f W e s t e r m a n n ( 1 9 8 5 ) . Briefly, the fixed gels w e r e rinsed in distilled water and then immersed overnight at r o o m temperature in 2 0 volumes o f a b s o lute a c e t o n e containing 2 0 % diphenyloxazole ( P P O ) . T h e dehydrated gels w e r e then e x p o s e d directly unto either Agfa safety curix RP1 or Hyperfilm-lsmax (Amer- s h a m ) films.
RESULTS
EFFECT OF ULTROSER-G ON THE SURVIVAL OF NODULAR MF IN VITRO
T o investigate the effect o f Ultroser-G o n the in vitro survival o f nodular mf, as a substitute o f normal human serum, three concentrations o f the product w e r e used.
As s h o w n in figure 1, the in vitro culture o f nodular m f could conveniently b e performed in medium contai
ning Ultroser-G at either 1 or 2 % o f this product. T h e viability o f the nodular m f was found to d e c r e a s e to less than 4 0 % after 4 8 hours in medium supple
m e n t e d with either serum or Ultroser-G. After the s a m e incubation time the viability was less than 2 0 % in medium RPMI 1 6 4 0 only (Fig. 1 ) . It should b e n o t e d that the viability o f the nodular mf in medium SLipplemented with 1 % ( v / v ) Ultroser-G was found to a p p r o a c h that in NHS in the early periods o f the cul
tures (within 24 hours). H e n c e , this concentration o f the synthetic product and a total incubation period o f 24 hours were used in our subsequent experiments and could as well b e r e c o m m e n d e d for prolonged in vitro m a i n t e n a n c e o f parasites in serum-free media.
EFFECT OF INCUBATION TIME ON THE SYNTHESIS AND RELEASE OF ADULT FEMALE E S P
In order to determine the optimal conditions for the in vitro synthesis and release o f radiolabelled parasite m o l e c u l e s with time, the effect o f FCS, NHS, NRS and the serum substitute Ultroser-G o n the synthesis and release o f adult female ESP, was investigated as a func
tion o f the amount o f TCA-precipitable radioactivity.
D u e to the difficulty in obtaining large numbers o f living adult female worms, e a c h assay was performed with o n e adult female w o r m only, with the six worms b e i n g o b s e r v e d simultaneously. T h e viability o f the
Parasite, 1997, 4, 351-358
3 5 3 Mémoire
Fig. 1. — Effect of Ultroser-G on the survival of O. volvulus nodular microfilariae. Nodular mf were cultured in medium RPMI 1640 only (RPMI) or this medium supplemented with either 10 % human serum (HS) or Ultroser-G at 1 % (1 % UTS), 2 % (2 % UTS) and 4 % (4 % UTS). The viability of the worms was assessed over a 72 hours period under sterile conditions and at room temperature.
parasites during the entire period o f the experiment was carefully m o n i t o r e d by both visual and micro
s c o p i c m o v e m e n t s o f the worms. All the w o r m s w e r e found to b e viable at the e n d o f the 24-hour incuba
tion. T h e results depicted in figure 2 indicate that the amount o f TCA-precipitable radioactivity in the adult female cultures containing the sera or Ultroser-G was influenced by the nature o f the medium supplement and the time after addition o f the radiolabelled methio
nine. Although the overall trend was similar in all the assays, the rate o f synthesis and h e n c e release o f de
novo synthesized parasite polypeptides a p p e a r e d to b e rapid within the first six hours following their s e l e c tive starvation from the precursor a m i n o acid. Pro
l o n g e d in vitro c u l t u r e o f t h e p a r a s i t e s ( b e y o n d 6 hours) led to gradual d e c r e a s e in the amounts o f the TCA-precipitable ESP (Fig. 2 ) . With the e x c e p t i o n o f NHS, all other supplements displayed rather similar rates o f ESP.
S D S - P A G E PATTERNS
O F T H E IN VITRO RELEASED P R O D U C T S ( E S P )
In the preliminary experiments, adult female w o r m s and nodular m f w e r e labelled in m e d i u m s u p p l e m e n t e d with FCS. W h e n the culture supernatant w e r e analyzed by SDS-PAGE and autoradiography, at least 3 0 polypeptides with molecular weights ranging bet
w e e n 7.0 and 174 kDa appeared to b e synthesized and released in vitro as early as 2 hours after addition o f the radioactive m e t h i o n i n e (data not s h o w n ) .
W h e n the nodular m f and adult female in vitro released products g e n e r a t e d in culture media s u p p l e m e n t e d with the different sera or Ultroser-G w e r e similarly ana
lyzed, the polypeptide patterns o f the [3 5S]-methionine labelled ESP from adult females (Fig. 3 ) and nodular m f (Fig. 4 ) w e r e as s h o w n . For both parasite stages, the synthesis and release o f ESP u n d e r these condi
tions a p p e a r e d to b e most effective in m e d i u m contai
ning FCS and least in that containing NHS. O t h e r sup
plements such as heat-inactivated NRS and Ultroser-G a p p e a r e d to b e equally effective.
Although the gel patterns o f the ESP w e r e similar in both stages, significant quantitative and qualitative dif
ferences in the released polypeptides w e r e o b s e r v e d with respect to the m e d i u m supplement. T h u s adult female w o r m s p r o d u c e d stable high molecular weight polypeptides w h e n cultured in m e d i u m s u p p l e m e n t e d with FCS and not in that containing either NHS, NRS or Ultroser-G (Fig. 3 ) . Nodular m f o n the other hand p r o d u c e d identical p o l y p e p t i d e patterns w h e n cul
tured in media s u p p l e m e n t e d with either FCS, NRS or Ultroser-G (Fig. 4 ) . Furthermore, the d e t e c t e d c o m p o nents were present in the labelled somatic extracts from nodular m f and in vitro released eggs (Fig. 4, lanes 7 and 8 respectively).
S A K W E A.M., E V E H E M . - S . B . & TITANJI V.P.K.
E X C R E T O R Y / S E C R E T O R Y PRODUCTS оf О. VOLVULUS
Fig. 2. — Effect of incubation time on the in vitro synthesis and release of [3 5S]-methionine labelled proteins of adult female worms. One adult female worm per assay was labelled in methionine-free medium supplemented with either normal human serum (NHS), heat-inac
tivated normal rabbit serum (NRS), Ultroser-G (UTS), normal human serum incubated at 37 °C (NHS at 3 7 ° C ) , fetal calf serum with heat- shock treatment of parasites (HSR) and fetal calf serum (FCS). Aliquots of the culture supernatant from each of the assays were TCA-pre- cipitated and analyzed by liquid scintillation counting.
Fig. 3- — Major in vitro released polypeptides of O. volvulus adult female worms. One adult female worm per assay was labelled in methionine-free medium supplemented with either NHS (lane 1), NRS (lane 2), Ultroser-G (lane 3), FCS with heat-shock treatment of the parasites (lane 4 ) , FCS (lane 5) or NHS at 37 °C (lane 6) and aliquots of the culture supernatant analyzed by SDS-FAGE and autoradiography.
Fig. 4 . — Major in vitro released polypeptides of O. volvulus nodular microfilariae. Nodular mf cultures containing 6.000 to 10.000 worms per assay were labelled in methionine-free medium supplemented with either NHS at 37 °C (lane 1), NRS (lane 2), Ultroser-G (lane 3).
FCS after heat shock treatment (lane 4 ) , FCS at 37 °C (lane S) or FCS (lane 6 ) . Aliquots of the culture supernatant or somatic extracts from labelled nodular infilane 7 ) and in vitro released eggs (lane 8 ) were then analyzed by SDS-PAGE and autoradiography.
3 5 5 Parasite, 1997, 4, 351-358
Mémoire
S A K W E A.M., E V E H E M.-S.B. & TITANJI V.P.K.
H e a t - s h o c k treatment o f the parasites at 4 2 °C for 20 min followed b y labelling a n d c o n t i n u e d incuba
tion at 37 °C had little or n o stimulatory effect o n the synthesis a n d / o r shedding o f proteins from nodular m f (Fig. 4 , l a n e 4 ) . U n d e r t h e s a m e c o n d i t i o n s adult females w e r e a b l e to release a 6 3 k D a polypeptide (Fig. 3, lane 4 ) .
REACTIVITY OF THE IN VITRO RELEASED PRODUCTS WITH HOST ANTIBODIES
In order to d e t e r m i n e w h e t h e r the s e c r e t e d proteins w e r e antigenic to t h e host, the ESP from the t w o worm stages prepared in media supplemented with the sera or Ultroser-G w e r e immunoprecipitated with a h y p e r - i m m u n e patient serum pool (n = 5 4 ) a n d a control NHS p o o l (n = 1 0 ) . As s h o w n in figure 5, at least 14 o f t h e s e c r e t e d polypeptides from nodular m f maintained in medium with either NRS or Ultroser-G, a p p e a r e d to react with t h e patient antibodies. Under similar conditions t w o o f the polypeptides with m o l e cular weights o f 7.0 a n d 1 7 4 k D a w e r e found to react
with the normal h u m a n serum proteins (Fig. 5, lane 3 ) . Similar results w e r e o b t a i n e d with the ESP from the adult female cultures (Fig. 6 ) .
T h e r e w a s also apparent similarity in the s e c r e t e d anti
g e n s a n d c o m p o n e n t s with m o l e c u l a r weights o f 174, 68, 3 3 , 16 a n d 1 3 k D a w e r e prominently s e c r e t e d b y both stages. T h e 6 3 k D a antigen from the adult female- w o r m s w h i c h w a s s e c r e t e d / e x c r e t e d after heat-shock treatment a p p e a r to b e an adult female stage specific antigen. W h e n t h e ESP from both stages w e r e preli
minarily cleared with NHS before immunoprecipitation, n o d e t e c t a b l e bands c o u l d b e s e e n o n t h e autoradio
grams e v e n after p r o l o n g e d e x p o s u r e times (data not s h o w n ) .
Fig. 5. — Excretory/secretory antigens of O. volvulus nodular mf.
Nodular mf ESP prepared from parasites labelled in methionine-free medium supplemented with NRS (lane 1) and Ultroser-G (lane 2) were immunoprecipitated with a hyper-immune patient serum pool (и = 54) and the immune complexes analyzed by SDS-PAGE and autoradiography. Nodular mf ESP generated from parasites cultured in medium supplemented with FCS were used to precipitate the normal human control serum pool (lane 3).
Fig. 6. — Excretory/secretory antigens o f O. volvulus adult female worms. One adult female worm per assay was labelled in methio
nine-free medium supplemented with either NHS (lane 1), NRS (lane 2), Ultroser-G (lane 3), FCS with heat shock treatment (lane 4 ) , NHS at 37 °C (lane 5) or FCS (lane 6). Aliquote of the culture super
natant (ESP) were immunoprecipitated with a hyper-immune patient serum pool and the immune complexes analyzed by SDS-PAGE and autoradiography. Adult female ESP generated from medium sup
plemented with FCS were used to precipitate the control NHS pool (lane 7 ) .
DISCUSSION
We have successfully used the synthetic pro
duct Ultroser-G as a serum substitute for the in vitro maintenance of O. volvulus adult females and nodular mf. The viability of the para
sites in medium supplemented with 1 % Ultroser-G as well as the proteins and antigens released in vitro by these life-cycle stages were similar to those obtained by culturing the two life-cycle stages in the presence of serum.
E X C R E T O R Y / S E C R E T O R Y PRODUCTS O F 0 .
voimus
Several studies h a v e d e m o n s t r a t e d the i m p o r t a n c e o f h u m a n serum for p r o l o n g e d in vitro m a i n t e n a n c e o f O. volvulus ( E n g e l b r e c h t & Shulz-Key, 1 9 8 4 ; Walter, 1 9 8 8 ) . H o w e v e r , h u m a n s e r u m e s p e c i a l l y in t h e e n d e m i c areas has b e c o m e a risky reagent for this a n d o t h e r applications d u e to the s p r e a d o f HIV/AIDS a n d hepatitis infections. T h e present investigation d e m o n s trates that m e d i u m s u p p l e m e n t s s u c h as fetal c a l f s e r u m ( F C S ) , h e a t - i n a c t i v a t e d n o r m a l rabbit s e r u m (NRS) a n d Ultroser-G c a n also facilitate p r o l o n g e d in vitro survival o f this c o n n e c t i v e tissue-dwelling para
site a n d m o r e o v e r , p r o m o t e the active synthesis a n d release o f parasite products.
T h e viability o f the parasites stages u s e d in this study w a s carefully m o n i t o r e d b y b o t h visual a n d micro
s c o p i c m o v e m e n t s . Adult parasites w e r e found to b e viable throughout the e x p e r i m e n t s while the viability o f nodular m f d e c r e a s e d from m o r e than 9 5 % to about 75 % . It w a s therefore e x p e c t e d that only c o m p o n e n t s labelled de novo c o u l d b e d e t e c t e d b y the m e t h o d s d e s c r i b e d in this study. F o l l o w i n g preliminary e x p e r i m e n t s to d e t e r m i n e t h e optimal conditions, the effect o f t h e s e s u p p l e m e n t s o n protein synthesis a n d s e c r e tion w a s m o n i t o r e d as d e s c r i b e d in materials a n d m e t h o d s . Further investigations o n t h e s e c r e t e d m o l e cules w e r e nevertheless limited to their sizes (Figs. 3 a n d 4 ) and antigenicity (Figs. 5 a n d 6 ) . This not
withstanding, the effect o f these s u p p l e m e n t s o n in vitro protein synthesis a n d s e c r e t i o n (Fig. 2 ) suffi
ciently r e p r o d u c e s the characterization o f the s e c r e t e d proteins, as s i x w o r m s w e r e o b s e r v e d simultaneously.
Moreover, individual m e d i u m s u p p l e m e n t s h a v e sub
sequently b e e n u s e d to culture adult w o r m s separa
tely although radiolabelling c o u l d not b e d o n e in these c a s e s (results n o t illustrated)
T h e e x c r e t o r y / s e c r e t o r y products ( E S P ) derived from parasites cultured in the p r e s e n c e o f these supplements w e r e found to b e immuno-reactive with patient s e r u m a n t i b o d i e s , t h e r e b y indicating that in vitro mainte
n a n c e o f t h e s e parasite stages in the p r e s e n c e o f t h e s e s u p p l e m e n t s did not c o m p r o m i s e the antigenicity o f the s e c r e t e d / e x c r e t e d m o l e c u l e s . This finding also sug
gests that in vitro m a i n t e n a n c e o f t h e parasite stages as such is a reliable method o f obtaining relatively pure and functionally important ESP suitable for immuni
zation o f rabbits or o t h e r e x p e r i m e n t a l animals. T h e resulting antibodies w o u l d b e e x p e c t e d to b e void o f c o n t a m i n a t i n g a n t i b o d i e s to h u m a n s e r u m c o m p o n e n t s w h i c h c o u l d b e g e n e r a t e d if t h e ESP w e r e pre
pared from medium s u p p l e m e n t e d with h u m a n serum.
Active protein synthesis and release a p p e a r e d to b e o b s e r v e d up to six hours after addition o f the radio
active precursor in all the assays (Fig. 2 ) , indicating that a c h a n g e in the culture m e d i u m or the r e p l e n i s h m e n t o f the c o m p o n e n t s o f t h e m e d i u m b e c a m e n e c e s s a r y .
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The d e t e c t e d ESP w e r e as well d e t e c t e d in the somatic extracts o f nodular m f a n d in vitro r e l e a s e d e g g s (Fig. 4 ) , p r o b a b l y indicating that most o f the secreted c o m p o n e n t s c o u l d b e o f s o m a t i c origin ( H a q u e &
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ACKNOWLEDGEMENTS
T
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WAITER
