P A R A S I T E B I O L O G Y A N D B I O C H E M I S T R Y
REPRODUCTIVE B I O L O G Y A N D P O P U L A T I O N DYNAMICS O F ONCHOCERCA VOLVULUS I N T H E V E R T E B R A T E H O S T
S C H U L Z - K E Y H.* & S O B O S L A Y P.T.*
KEYWORDS : Onchocerca volvulus, worm load, fecundity, turnover of microfila
riae.
I
n filariases microfilariae play the k e y role for the transmiss i o n o f the parasite a n d the d e v e l o p m e n t o f p a t h o l o g y . Therefore, various a s p e c t s o f the parasites' reproductive b i o logy and population dynamics in the host are prime c o n s i d e r a t i o n s for t h e c o n t r o l o f f i l i a r i a s e s . S i n c e m i c r o f i l a r i a e r e l e a s e d from female w o r m s are not discharged, but a c c u mulate and survive in the host for a c o n s i d e r a b l e time, the reproduction o f filarial w o r m s is s u g g e s t e d to b e regulated to a greater d e g r e e by intrinsic host factors. T h e s e may, h o w e ver, vary during the c o u r s e o f infection and d e p e n d o n the r e s p o n s e o f the individual host. S i n c e there is n o pulmonary microfilarial r e s e r v o i r in Onchocerca i n f e c t i o n s , different regulatory m e c h a n i s m s are s u g g e s t e d for s p e c i e s with b l o o d - and skin-dwelling microfilariae (Schulz-Key, 1 9 8 8 ) .
DEVELOPMENT AND HABITAT OFADULT WORMS
I
nfective larvae ( L 3 ) moult to the fourth stage within 3-7 days, and a final moult to b e the juvenile w o r m s o c c u r s several w e e k s later. Immature w o r m s s e e m to b e attracted to existing nodules, m a y settle o n their surface and form satellite o r c o m p o s i t e n o d u l e s . T h u s , y o u n g , o l d and calcified d e a d w o r m s are often found associated in the s a m e nodule (Schulz-Key, 1 9 8 8 ) .
O n c h o c e r c o m a t a are found o n distinct sites o f predeliction o f the host and their a c c u m u l a t i o n m a y h e l p the s e x e s fo find e a c h other. O n average, 1-2 m a l e and 2-3 female w o r m s c a n b e f o u n d d i s t r i b u t e d in 8 0 % o f t h e n o d u l e s . Accumulation o f m o r e than 5 0 w o r m s c a n occur, but this is the e x c e p t i o n . In contrast to the sessile females male w o r m s r e g u l a r l y l e a v e t h e n o d u l e s - p r e s u m a b l y in s e a r c h o f females. C o n s e q u e n t l y , in e x c i s e d o n c h o c e r c o m a t a a striking p r e d o m i n a n c e o f female w o r m s c a n b e o b s e r v e d o w i n g to m a l e w o r m s just m i g r a t i n g in t h e h o s t . T h e w a n d e r i n g impulsion and thus the reproductive activity o f male w o r m s is suggested to d e c r e a s e with a g e ( K a r a m et al, 1 9 8 7 ) . Most male w o r m s in o v e r a g e d w o r m populations, e.g. as found in t h e late p h a s e o f t h e O n c h o c e r c i a s i s C o n t r o l p r o g r a m m e ( O C P ) in W e s t Africa, are inactive and separated from n o n gravid females although associated in the s a m e nodule.
initiation o f m a l e s to m a t e . C o n v e r s e l y , o o c y t e s are r e l e a s e d from the n a r r o w o v a r i e s into the w i d e l u m e n o f the uteri i n d e p e n d e n t from t h e p r e s e n c e o f m a l e w o r m s . In c a s e female w o r m s d o not m a t e , large n u m b e r s o f d e g e n e r a t i n g a n d s k r i n k i n g o o c y t e s m a y a c c u m u l a t e w h i c h a r e l a t e r r e s o l v e d . T h e d e v e l o p m e n t o f an o o c y t e to a mature m i c r o filariae is e s t i m a t e d to t a k e n o t m o r e t h a n t h r e e to four w e e k s ( S c h u l z - K e y a n d Karam, 1 9 8 6 ) . Fecundity starts after a pre-mature p e r i o d o f n i n e to t w e l v e m o n t h s ( D u k e , 1 9 8 0 ; Schulz-Key, 1 9 8 8 ; S o b o s l a y et al, 1 9 9 1 ) .
DISTRIBUTION OF EMBRYONIC STAGES IN FEMALE WORMS
M
ature f e m a l e w o r m s o f s p e c i e s with b l o o d - d w e l l i n g microfilariae w e r e found to c o n t a i n e m b r y o n i c stages a n d microfilariae in b o t h b r a n c h e s o f the uteri throughout the patent infection ( M o s s i n g e r a n d W e n k , 1 9 8 6 ; M o s s i n g e r a n d B a r t h o l d , 1 9 8 7 ) . T h e i r e m b r y o g r a m s s h o w e d r a t h e r h o m o g e n e o u s patterns indicating that uterine microfilariae w e r e p e r m a n e n t l y p r e s e n t a n d c o n t i n u o u s l y r e l e a s e d into t h e h o s t r e f l e c t i n g a r a p i d t u r n o v e r o f t h e m i c r o f i l a r i a l p o p u l a t i o n .In c o m p a r i s o n , f e m a l e O. volvulus s h o w a m o r e h e t e r o g e n e o u s distribution o f uterine stages. Less than t w o third o f the adult females c o n t a i n e m b r y o n i c stages a n d microfila
riae, a l t h o u g h primary o o c y t e s are p r e s e n t a t t a c h e d to the rachis in the distal o v a r i e s in all o f them. O n e third, or in o v e r a g e d w o r m p o p u l a t i o n s distinctly m o r e , s h o w o o c y t e s or e m p t y uteri, e v e n in big n o d u l e s w h e n a s s o c i a t e d with a sufficient n u m b e r o f m a l e s . T h i s typical distribution pattern o f varying reproductive p h a s e s is i n d e p e n d e n t from the a g e o f the w o r m s and the adult w o r m load.
T h e p h a s e o f transient reproductive "inactivity" s e e m s to b e rather short. O o c y t e s and early d e v e l o p m e n t a l stages o f a n e w batch can b e o b s e r v e d in female w o r m s which still har
b o u r s o m e degenerating microfilariae, but n o further precur
sor stages o f the latter. In addition, the analysis o f w o r m s isolated from patients treated with d a i g s that interfere with the intrauterine d e v e l o p m e n t lead to the c o n c l u s i o n that the reproduction o f O. volvulus o c c u r s in a s y n c h r o n o u s b a t c h e s lasting t w o to four m o n t h s e a c h ( S c h u l z - K e y a n d Karam, 1 9 8 7 ) . Similar r e p r o d u c t i o n patterns c a n b e o b s e r v e d for Onchocerca ochengi ( T r e e s et al, 1 9 9 2 ) , Onchocerca gihsoni ( V a n k a n and C o p e m a n , 1 9 8 8 ) , Onchocerca s p e c i e s in wild animals (Schulz-Key, 1 9 8 3 ) . T h e y might b e typical for s p e c i e s with skin-dwelling microfilariae and might play a role in the microfilarial population dynamics in the host.
MATING, INSEMINATION AND FERTILIZATION
T
h e stimuli a n d p h e r o m o n s w h i c h attract m a l e s to the f e m a l e s in the n o d u l e s are u n k n o w n . S i n c e s p e r m a t o z o a a r e s c a n t y in f e m a l e s with e m p t y uteri, s h e d d i n g o f o o c y t e s into t h e uteri s e e m s to b e a prerequisit for the sti-* Institute of Tropical Medicine, University of Tubingen, Germany
REPRODUCTIVE CAPACITY AND MICROFILARIAL RELEASE
IN VIVOh e r e p r o d u c t i v e c a p a c i t y o f f e m a l e w o r m s c a n b e I a s s e s s e d b y e m b r y o g r a m s w h i c h quantify the n u m b e r o f intrauterine stages actually present. T h i s "snapshot" c a n -
ParasJte, 1994, 1, I S 53
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/199401s1053
PARASITE B I O L O G Y AND BIOGI [EMISTRY
not, h o w e v e r , indicate h o w m a n y microfilariae p e r d a y a r e p r o d u c e d o r r e l e a s e d . O b s e r v a t i o n s o n w o r m s m a i n t a i n e d in vitro o r e x c i s e d from patients at different p e r i o d s after c h e m o t h e r a p y s u g g e s t that a b o u t 7 0 0 t o 1,500 microfilariae p e r f e m a l e a r e r e l e a s e d into t h e host o n a v e r a g e p e r d a y , i.e. o n l y a portion o f the microfilariae actually d e v e l o p e d in utero l e a v e t h e f e m a l e w o r m s ( S c h u l z - K e y , 1 9 9 0 ) . T h e daily output is a n o r d e r t o m a g n i t u d e l o w e r than that o b s e r v e d for s p e c i e s with b l o o d - d w e l l i n g microfilariae. F o r e x a m p l e , f e m a l e s o f Loa, Litomosoides o r Dirofilaria e x p e l 1 0 , 0 0 0 t o 2 0 , 0 0 0 m i c r o f i l a r i a e i n t o t h e h o s t d a i l y ( E b e r h a r d a n d Orihel, 1 9 8 8 ; H a w k i n g , 1 9 5 4 ; M o s s i n g e r a n d W e n k , 1 9 8 6 ; W e i n s t e i n a n d S a w y e r , 1 9 6 1 ) .
In contrast t o o t h e r filarial s p e c i e s microfilariae o f O. volvu
lus a r e n o t e x p e l l e d b y t h e female w o r m b u t l e a v e it acti
vely o n e b y o n e . It t a k e s at least 5 t o 1 0 s e c o n d s for a microfilaria t o l e a v e t h e f e m a l e w o r m w h e n it h a s arrived at the vulva. T h i s is o b v i o u l s y a limiting factor. Microfilariae w h i c h stay in t h e uteri gradually d e g e n e r a t e a n d a r e later o n r e s o r b e d ( S c h u l z - K e y , 1 9 8 8 ) .
THE REPRODUQIVELIFESPAN OF O^VOLVULUS
I
h e r e p r o d u c t i v e lifespan h a s b e e n e s t i m a t e d b y longitudinal skin s n i p surveys during 13 t o 14 years o f s u c c e s s f u l v e c t o r c o n t r o l in t h e O C P a r e a i n W e s t A f r i c a (Plaisier et al., 1 9 9 1 ) . It w a s c o n c l u d e d that t h e r e p r o d u c tive lifespan o f t h e s a v a n n a strain o f t h e parasite lies b e t w e e n 9 a n d 11 y e a r s . T h i s i n d i r e c t a s s e s s m e n t o f t h e r e p r o d u c t i v e lifespan is in a c c o r d a n c e w i t h o b s e r v a t i o n s m a d e b y direct analysis o f o n c h o c e r c o m a t a from o n c h o c e r c i a s i s p a t i e n t s in o t h e r a r e a s o f t h e c o n t r o l p r o g r a m m e ( K a r a m et al, 1 9 8 6 ) .
REGULATION OF REPRODUCTION
T
h e m e c h a n i s m s that r e g u l a t e microfilarial p r o d u c t i o n and r e l e a s e a r e u n k n o w n a n d s e e m t o b e multifactorial.It is r a t h e r u n l i k e l y that t h e r e l e a s e o f m i c r o f i l a r i a e is m e c h a n i c a l l y regulated b y t h e f e m a l e w o r m s , e.g. b y t h e s p h i n c t e r o f the vulva. T h e u n p a i r e d part o f t h e uterus m a y b e e m p t y o r s p a r s e l y p o p u l a t e d , e v e n w h e n sufficient h e a - vely wriggling microfilariae are p r e s e n t in a posterior s e c tion o f t h e uterus, p e r h a p s in e x p e c t a n c e o f a stimulus for active e v a s i o n . O b s e r v a t i o n s o n patients treated with iver
m e c t i n a n o n v e r y o l d w o r m p o p u l a t i o n s in a r e a s w i t h i n t e r r u p t e d t r a n s m i s s i o n s u p p o r t o u r h y p o t h e s i s that t h e p r o d u c t i o n a n d r e l e a s e o f intra-uterine microfilariae a r e par
tially o r e v e n p r e d o m i n a n t l y regulated b y rather i n d e p e n d e n t m e c h a n i s m s , s o m e o f t h e m i n t r i n s i c in t h e h o s t ( S c h u l z - K e y , 1 9 9 0 ) .
In o v e r a g e d w o r m p o p u l a t i o n s , e.g. in i n h a b i t a n t s o f t h e OCP area, t h e t u r n o v e r o f microfilariae in t h e host is very low. An i n c r e a s e d n u m b e r o f f e m a l e w o r m s s h o w non-ferti
l i z e d o o c y t e s o r e m p t y uteri, a n d in g r a v i d f e m a l e s t h e n u m b e r o f d e v e l o p e m e n t u t e r i n e s t a g e s is l o w , m a n y o f t h e m p a t h o l o g i c a l l y altered. A l o n g lasting interruption o f transmission p r o v i d e d gradual c h a n g e s o f t h e i m m u n e res
p o n s e o f the h o s t at t h e s a m e time.
T h e o b s e r v e d d e c r e a s e o f parasite reproductivity s e e m s t o b e r e v e r s i b l e (Awadzi et al., 1 9 8 5 ) , a n d therefore s o m e o f
t h e s e c h a n g e s might b e c o r r e l a t e d with t h e r e g u l a t i o n o f the parasitdermia. Interestingly, uterine microfilariae p r o d u c e d in w o r m s o f i n h a b i t a n t s w h o h a v e a l r e a d y b e c o m e n e g a t i v e in skin snips a r e n o m o r e r e l e a s e d a n d d e g e n e r a t e in hypergravid female w o r m s ( S c h u l z - K e y et al., 1 9 8 7 ) . T h i s o b s e r v a t i o n further s u p p o r t s o u r h y p o t h e s i s o f i n d e p e n d e n t m e c h a n i s m s o f microfilaria p r o d u c t i o n a n d r e l e a s e .
SKIN MICROFILARIAE AND ADULT WORM LOAD
h e correlation b e t w e e n t h e adult a n d microfilaria l o a d is varying from p e r s o n t o p e r s o n d e p e n d i n g o n t h e res
p o n s e o f t h e individual host. T h e r e a r e several indications that b o t h t h e a c c u m u l a t i o n o f adult O. volvulus in t h e host and t h e microfilarial densities a r e not linearly e s t a b l i s h e d . T h e r e s e e m s t o b e an increasing resistance t o s u p e r i n f e c tions, as h a s b e e n already s u g g e s t e d for s o m e o t h e r filarial i n f e c t i o n s ( B a r t h o l d a n d W e n k . 1 9 9 2 ; D a y et al.. 1 9 9 1 ) , a n d m i c r o f i l a r i a l d e n s i t i e s a r e c o n t r o l l e d b y t h e h o s t ' s i m m u n e s y s t e m as well. T h e s e are major restrictions for a n y c a l c u l a t i o n a n d estimation.
In a h y p e r e n d e m i c village o f t h e Liberian rain-forest, t h e micro- a n d macrofilarial loads w e r e a s s e s s e d b y skin snips and n o d u l e c t o m i e s (Albiez et al., 1 9 8 4 ) . All palpable n o d u l e s o f 117 inhabitants w e r e r e m o v e d , t h e w o r m loads a n a l y s e d and correlated with t h e m e a n microfilarial load, w h i c h w a s 1 2 x l 06 microfilaria in e a c h patient o n average ( S c h u l z - K e y , 1 9 9 0 ) . A m e a n lifespan o f a microfilaria o f 1.0 t o 1.5 years anticipated a stable parasitdermia n e e d s t o r e p l a c e 2 2 - 3 3 x 1 0 3 microfilariae every day. B a s e d o n t h e a b o v e estimated daily release o f microfilariae a p p r o x i m a t e l y 3 0 females are n e e d e d to p r o d u c e t h e s e n u m b e r s o f microfilariae. Although an unk
n o w n proportion o f deep-laying nodules w a s not a c c e s s i b l e for e x t i r p a t i o n , o u r a s s e s s m e n t o f a m e a n n u m b e r o f 1 6 f e m a l e s ( g e o m . m e a n ) p e r p a t i e n t f o u n d in t h e e x c i s e d nodules signals a right order t o magnitude in o u r calculation.
ACKNOWLEDGEMENTS
ur investigations w e r e s u p p o r t e d b y t h e C o m m i s s i o n o f the E u r o p e a n C o m m u n i t i e s ( S T D 1-3), W H O ( T D K ) and t h e G e r m a n A s s o c i a t i o n o f T e c h n i c a l C o o p e r a t i o n ( g t z ) .
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ISOLATION O F N E W M A R K E R S T O DETECT GENETIC VARIATION I N ONCHOCERCA VOLVULUS
HERDER S.*, BELLEC CH* AND CUNY G.**
KEYWORDS : Onchocerca volvulus, genetic variability. RAPD. microsatellite.
PCR.
SUMMARY
Newly developed techniques (RADP : Random Amplified Polymorphic DNA and microsatellite DNA sequences) were used to study genetic
* Laboratoire d'Epidemiologie de Maladies ä Vecteurs, Centre ORS- TOM d e M o n t p e l l i e r , 9 1 1 a v . A g r o p o l i s , BP 5 0 4 5 , 3 4 0 3 2 Montpellier, France.
** Laboratoire Retrovirus-Parasites, Centre ORSTOM de Montpellier, 911 av. Agropolis. B P 5045. 34032 Montpellier. Fiance.
variation in Onchocerca volvulus. RAPD technique, derived from the Polymerase Chain Reaction (PCR), allow clear distinction between the different species of the genus Onchocerca.
Microsatellite DNA has been shown to be useful as polymorphic mar
kers for populations and even for Individuals. Short repeated sequences (CA repeats) have been isolated, and a PCR assay using such microsatellite DNA sequences to generate polymorphisms Is cur
rently being experimented.
In W e s t A f r i c a , p a r t i c u l a r l y i n t h e O C P a r e a ( O n c h o c e r c i a s i s Control P r o g r a m m e ) , at least t w o strains o f the parasite a r e k n o w n t o exist. T h e s o - c a l l e d "savannah'' strain a s s o c i a t e d with high b l i n d n e s s level, a n d t h e "forest"
s t r a i n a s s o c i a t e d w i t h a m i l d e r f o r m o f o n c h o c e r c i a s i s w h i c h c a u s e s b l i n d n e s s in less than 1 % o f t h e population (Prost et al., 1 9 8 0 ) . H o w e v e r , Sierra L e o n e is e x c e p t i o n a l in t h a t h i g h l e v e l s o f b l i n d n e s s a r e f o u n d i n f o r e s t a r e a s ( M c M a h o n et al., 1 9 8 6 ) .
V a r i o u s Onchocerca specific DNA p r o b e s h a v e b e e n d e v e l o p e d (Meredith et al., 1 9 8 9 ) a n d also strain specific p r o b e s ( E r t t m a n n et al., 1 9 8 7 a n d 1 9 9 0 ) . T h e s e D N A p r o b e s s e q u e n c e s a r e b a s e d o n t h e 150 b p repeat family. Recently, s t u d i e s c o n d u c t e d in W e s t Africa h a v e s h o w n that DNA p r o b e c l a s s i f i c a t i o n c o r r e l a t e s with t h e e p i d e m i o l o g y o f b l i n d n e s s ( Z i m m e r m a n et al., 1 9 9 2 ) .
In C a m e r o o n , t h e situation is m o r e c o m p l e x , n o t a b l y t h e p a t h o l o g y o f b l i n d n e s s is distributed without apparent rela
tion t o b i o c l i m a t i c z o n e s ( D u k e , 1981 ; B o u s s i n e s q et al., 1 9 9 3 ) . T o date, n o studies similar t o t h o s e carried o u t in W e s t Africa h a v e b e e n d o n e in C a m e r o o n .
W e a r e investigating o t h e r m o l e c u l a r m a r k e r s that m a y b e u s e f u l f o r s t u d i e s o n g e n e t i c v a r i a t i o n in O. volvulus, n a m e l y RAPD a n d microsatellite s e q u e n c e s .
P o l y m o r p h i s m in g e n o m i c f i n g e r p r i n t s g e n e r a t e d b y R a n d o m Amplified P o l y m o r p h i c DNA ( R A P D ) is a recently d e v e l o p e d assay that is b a s e d o n t h e amplification b y t h e P o l y m e r a s e C h a i n R e a c t i o n ( P C R ) o f r a n d o m DNA frag
ments. In RAPD m a p p i n g , d e c a m e r o l i g o n u c l e o t i d e primers o f arbitrary' s e q u e n c e b u t with a G C c o n t e n t o f 5 0 % o r h i g h e r a r e u s e d t o a m p l i f y f r a g m e n t s o f g e n o m i c D N A (Williams etal. 1 9 9 0 ; W e l s h a n d McClelland, 1 9 9 0 ) . R A P D h a s b e e n s u c c e s s f u l l y a p p l i e d t o t h e a n a l y s i s o f g e n o m i c DNA variation o f several o r g a n i s m s (Williams et al, 1 9 9 0 ; Hadrys et al, 1 9 9 2 ; Klein-Lankhorst et al., 1991 ; Crowhurst et al.. 1991 ; Mazurier et al.. 1 9 9 2 ) . Strains c a n b e distinguished b y c o m p a r i n g p o l y m o r p h i s m s in g e n o m i c fingerprints ( W e l s h et al. 1 9 9 1 ) .
T h e particular a d v a n t a g e s o f RAPD t e c h n o l o g y is that small a m o u n t s ( n a n o g r a m s ) o f g e n o m i c DNA a r e n e e d e d a n d , u n l i k e PCR, n o prior s e q u e n c e k n o w l e d g e is n e e d e d . Microsatellite DNA s e q u e n c e s are short (from 2 t o 5 b p ) , tan- demly repeated s e q u e n c e s that have b e e n s h o w n t o b e use
ful a s p o l y m o r p h i c m a r k e r s for p o p u l a t i o n s a n d e v e n for individuals. T h e y have b e e n reported from a large panel o f eukaryotic s p e c i e s such as human, m o u s e , cattle, w h a l e s a n d insects (Tautz, 1 9 8 9 ; Love etal. 1 9 9 0 ; Swaiger etal, 1 9 9 2 ; W e b e r a n d May, 1 9 8 9 ; H u g h e s a n d Queller, 1 9 9 3 ) . T h e s e d i n u c l e o t i d e r e p e a t s a r e referred t o as microsatellites (Litt and Luty, 1 9 8 9 ) . Most o f t h e s e regions are less than 2 0 0 b p in length a n d PCR is used t o detect length polymorphisms.
W e d e s c r i b e h e r e t h e u s e o f t h e RAPD assay for t h e d e t e c tion o f variation in t h e g e n u s Onchocerca, using a set o f 2 0
