R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 1 8 ( 4 ) : 2 5 1 - 2 5 5 , O u t - D e z , 1 9 8 5
KINETICS OF THE
C E R C A R I A - S C H I S T O S O M U L U MTRANSFORMATION
I N V I V O :
2. THE EFFECT OF OXAM NIQUINE
A. L. M e lo
1and L.H. P ereira
1T o s t u d y t h e c e r c a r i a - s c h i s t o s o m u l u m t r a n s f o r m a t i o n in vivo, u n d e r t h e i n f l u e n c e o f a n a n t i s c h i s t o s o m a l c o m p o u n d ( o x a m n i q u i n e ) , a m o d e l u s i n g c e r c a r i a l i n j e c t i o n s i n t o t h e a b d o m i n a l c a v i t y o f m i c e w a s c h o s e n . T h i s p r o c e d u r e p r o v i d e d e a s y a n d r e p r o d u c i b l e r e c o v e r i e s o f l a r v a e f r o m p e r i t o n e a l w a s h i n g s w i t h a p p r o p r i a t e s o l u t i o n s f o r a l o n g t i m e ( 3 0 t o 1 8 0 m i n ) a f t e r i n o c u l a t i o n . T h e r e s u l t s s h o w t h a t h i g h d o s e s o f o x a m n i q u i n e ( g i v e n i n t r a m u s c u l a r l y o n e h o u r b e f o r e t h e i n f e c t i o n ) p r o d u c e a m a r k e d d e l a y i n t h e k i n e t i c s o f t h e c e r c a r i a - s c h i s t o s o m u l u m t r a n s f o r m a t i o n . C e r c a r i a e , t a i l - l e s s c e r c a r i a l b o d i e s a n d s c h i s t o s o m u l a w e r e r e c o v e r e d f r o m t h e p e r i t o n e a l c a v i t y o f d r u g t r e a t e d m i c e i n n u m b e r s s i g n i f i c a n t l y d i f f e r e n t f r o m t h o s e r e c o v e r e d f r o m u n t r e a t e d m i c e .
K e y words: S c h i s t o s o m a m a n s o n i . C ercariae. S ch isto so m u le. I n v i v o trans form ation. O xam n iq u in e.
In the ch em oth era p y o f sc h isto so m ia sis, oxam niquine has b ee n u sed for ab ou t 10 years b y oral adm inistration.
T h e co m p ou n d is a ctiv e intram uscularly at 2 0 0 m g /k g aga in st m ature s ch isto so m e s and yo u n g larvae in laboratory m ic e 8.
D ifferen t routes o f ex p erim en ta l in fection d o n ot alter the resu lts, and a ctivity again st early d ev elop in g forms o f the parasite has b ee n confirm ed after ex p o-sure o f m ouse skin to S c h i s t o s o m a m a n s o n i cercariae5, and after in ocu lation into the p erito n eal ca v ity o f m ice2 3 7 8 9 1 011 U sin g d o se s o f 6 0 to 4 0 0 m g/k g , on the transform ation o f cercariae into sch isto so m u la , no action w a s found b y P elleg rin o e t a l8. M e lo e t al2 show ed that d oses o f 1 ,0 0 0 and 2 ,0 0 0 m g/kg o f the drug produced so m e d ela y in th e transform ation p ro cess, 3 hours after cercarial inoculation. D o s e s o f 4 ,0 0 0 m g/kg o f oxam n iq u in e ca n a lso inhibit the lo ss o f the tail o f cercaria3.
T h e transform ation o f cercaria (a larva adapted to a freshw ater en viron m en t) to sch isto so m u lu m (an organism n ew ly adapted to h o st co n d itio n s) in vo lves several steps: o b ser v a tio n s o n that transform ation s, follow ing the intrap eritoneal in ocu lation o f ce rca r ia e4 provided so m e an sw ers to the q u estion . T h e p o ssib ility to discrim inate su ch step s i n v i v o , u sin g a n tisch is tosom al drugs, is attractive.
T h e purpose o f the p resen t w ork is to verify the activity o f oxam n iq u in e, at different d o se s and different tim es, on th e tran sform ation p ro cess o f cercariae into sch istosom u la.
1. D e p a r ta m e n to d e P a r a s ito lo g ia e G r u p o I n te r d e p a r ta m e n ta l d e E s tu d o s s o b r e E s q u is to s s o m o s e ( G I D E ) , I C B , U n iv e r s id a d e F e d e r a l d e M in a s G e r a is . C a ix a P o s ta l 2 4 8 6 , 3 0 0 0 0 B e lo H o r iz o n te , B ra sil.
T h is w o rk r e c e iv e d fin a n c ia l s u p p o r t fro m F I N E P , C N P q B ra z il, a n d fro m th e P a r a s itic D is e a s e s P r o g r a m m e . W H O - S w itz e r la n d .
R e c e b id o p a r a p u b lic a ç ã o e m 1 8 /3 /8 5
M A T E R I A L A N D M E T H O D S
T h e m eth od d escrib ed b y Pereira et a l10 to study the chem oprophylactic action o f com pounds on schisto som u la recovered from the peritoneal ca vity o f m ice w as used in ali experim ents. A lb in o m ice (m ale, weighing about 2 0 g) w ere in ocu lated intraperitoneally with
S c h i s t o s o m a m a n s o n i cercariae (L . E . strain), shed by laboratory-reared and in fected B i o m p h a l a r i a g l a -b r a t a (B e lo H o r izo n te strain). T h e organism s were con cen tra ted b y the m eth od o f P elleg rin o & M a c ed o 6, and 0 .5 ml o f w ell w ater, co n tain in g about 5 0 0 larvae, w ere injected b y a C o rn w a ll syringe w ith a 2 0 x 10 gauge n eed le.
A p propriate d o ses o f oxa m n iq u in e w ere m ixed w ith p o ly eth y len e g ly c o l in a mortar and injected (0.1 m l) intram uscu larly in the hind leg. T h e drug w as adm inistered o n e hour b efore the cercarial injections and d o se s o f 5 0 0 , 1 ,0 0 0 , 2 ,0 0 0 or 4 ,0 0 0 m g/k g w ere given to four groups o f five anim ais each . U n treated anim ais served as Controls.
A t different tim e intervals ranging from 30 m inutes to 3 hours after in ocu lation the m ice were k illed by cervica l fracture, and the p arasites recovered from the periton eal ca v ity w ith salin e. T h ey w ere then co n cen trated b y centrifugation and coun ted under a d issectin g m ic r o sc o p e 10.
T h e larvae w ere cla ssified initially as cercariae and ta iless organ ism s. T o separate cercarial bodies (w h ich rem ain a liv e in fresh w ater) from sch isto so m u la (w h ic h die in su ch co n d itio n s), 5 m l o f distilled w ater w a s dropped into a Petri dish containing the organism s. A fter 10 m in, th e liv e and d ea d larvae w ere recounted. In addition to the a b sen ce o f m otility, d ead larvae sh ow ed herniation o f acetabulum . T w o vital stains (neutral red and alizarin ) w ere added to the susp en sion o f organ ism s to ch a racterize the acetabular glands.
R E S U L T S
P
E
R
C
E
N
T
A
G
E
S
Melo AL, Pereira LH. Kinetics ofthe cercaria-schistosomulum transformation
in v ivo : 2 .The effect ofoxamniquine. Revista da
Sociedade Brasileira de Medicina Tropical 18: 251-255, Out-Dez, 1985
DEAD TAILLESS ORGAflISM S
SCHISTOSOM ULA
□
LIVING CERCARIAL B O 0IE SDEAD CERCARIAE
LIVING CERCARIAE
TREATEDÍSOO) CONTROLS TREATED (l.OOO) CONTROLS
too r
Melo AL, Pereira LH. Kinetics of the cercaria-schistosomulum transformation
in v ivo : 2.The effect of oxamniquine. Revista da
Sociedade Brasileira de Medicina Tropical 18: 251-255, Out-Dez, 1985
DEAD TAILLESS
ORGANISMS
□
L IV IN G CERCARIAL BODIESLIVING CERCARIAE
SCHISTOSOMULA E U DEAD CERCARIAE
T R E A T E D (2,000) CONTROLS t r e a t e d(4 ,o o o) ES
CONTROLS
zq m
Melo AL, Pereira LH. Kinetics of the cercaria-schistosomulum transformation
in vivo : 2 .The effectofoxamniquine. Revista da
Sociedade Brasileira de Medicina Tropical 18: 251-255, Out-Dez, 1985
w ere ob served b etw een larvae recovered from the control m ice and the anim ais treated w ith oxa m n iq u in e at 1 ,0 0 0 , 2 ,0 0 0 or 4 ,0 0 0 m g/kg.
P ercen tages o f livin g cercariae from larvae recovered from treated and control m ice w ere appro-priated chan ged to arc sin \ / x , and the S tu d en t’s t test w a s perform ed.
A s can b e seen in F igu re 1, the b eh aviou r o f experim ental groups (receivin g the drug at 5 0 0 m g/k g ) did not differ from the con trol group during the ob served tim e ( 3 0 to 1 8 0 m in u tes after cercarial in oculation). W ith 1 ,0 0 0 m g/k g, d ifferen ces w ere ob served b etw een the treated anim ais and Controls after 6 0 min. (p < 0 .0 1 ) and 9 0 (p < 0 .0 5 ) . A fter this tim e, the groups sh ow ed m arked differen ces in ali observations.
A n ev id en t d ela y in the transform ation p ro cess, w as found at different tim es o f ob servation , using 2 ,0 0 0 m g/k g o f oxa m n iq u in e (F ig . 2).
U sin g the d o se o f 4 ,0 0 0 m g/k g o f o x a m n i quine (F ig . 2 ), sh o w ed d ifferen ces in ali tim es, but abnorm al larvae w ere found: organism s presenting ch aracteristics o f both cercaria and sch isto so m u lu m , suggesting inhibition o f cercarial d ecaudation .
T h e use o f the vital stain to ob serve the effects o f oxam niquine on the acetab u lar glands did n ot sh ow marked differen ces. W h en alizarin or neutral red w ere u sed alm ost ali sch isto so m u la incorporated the vital stain, the colou r b ein g gen era lly in ten se (a s in the cercariae), in both groups.
D I S C U S S I O N
T h e stu dy o f cercaria-sch istosom u lu m trans form ation in the peritoneal ca v ity o f alb in o m ice, under the effects o f oxa m n iq u in e, w a s first carried o u t b y M elo et al^. H igh d o se s p roduced d ela y o f cercaria-sch istosom u lu m transform ation follow in g intraperi-toneal injection s o f cercarie in to m ice. C ercariae, tail-less cercarial b o d ies and sch isto so m u la w ere recovered (3 hours after the cercarial in ocu lation s) from the peritoneal c a v ity o f drug treated anim ais, in num bers significan tly different from th ose recovered from untreated m ice. S in c e untreated m ice induced transform ation o f alm o st ali th e injected cercariae, the data suggested that the co m p ou n d is active during the process o f h ost-larvae adaptation. A s ch a n ges u su ally start w ith the d etach m en t o f the tail fo llo w ed b y lo ss, rupture or ch a n ges o f the g ly c o ca lix in untreated m ice (M elo , 1 9 8 5 , u npu blished d ata), the step s cou ld be inhibited by high d o se s o f oxam n iq u in e.
In this w ork w e u sed again the in ocu lation o f cercariae into the periton eal ca v ity o f m ice , to fo llo w in detail the steps in vo lved in the Progressive adaptation o f cercariae to the vertebrate h ost under the effects o f oxam niquine. M o st cercariae reach the sch isto so -mular stage b etw een 9 0 - 1 2 0 m inutes after intraperi-toneal inocu lation in the control groups and in the m ice treated w ith 5 0 0 m g /k g o f oxa m n iq u in e. A t d o s e s o f
M e lo & P ereira3 sh ow ed that 4 ,0 0 0 m g/kg, oxa m n iq u in e inhibited cercariae d ecau d ation , but the Progressive lo s s o f g ly c o c a lix w a s not inhibited b y this com p ound.
In the presen t stu dy, the d ata su g g e st that the oxa m n iq u in e u sed at 1 ,0 0 0 , 2 ,0 0 0 and 4 ,0 0 0 m g/k g p roduces a d ela y in the tranform ation p ro cess. H igh num bers o f larvae recovered from treated m ice have n ot lo st their tail, and did n ot sh ow w ater in toleran ce, indicating a partial or total inhibition in the lo ss o f g ly c o c a lix .
O n th e other hand, it w as ob served that the d o se of 4 ,0 0 0 m g /k g inhibited the lo ss o f the tail sign ifican tly (p < 0 .0 1 ) . H o w ev e r, the oth er ch a n ges o f the cercaria to the sch isto so m u la r stage w ere o b served (lo s s o f g ly c o ca lix , w ater in toleran ce, w orm -like m o v e m en ts), su ggestin g that th ese ch a n g es m ay start w ith ou t the tail d etachm ent.
S om e authors112 b elieve that the loss o f the cerca rial tail is a m ere c o n se q u en c e o f m u scu lar activity o f the organ ism s. I f ox a m n iq u in e affects the cercariae b y reducing their m o v em en ts, the d etach m en t o f the tail w ou ld be less lik ely to occur.
T h e d ela y in the transform ation p ro cess, under an an tisch isto so m a l com p ou n d , co u ld b e ex-p lain ed b y an inhibitory m ec h a n ism o f tail lo ss or by alterations in the ca p a b ility o f lo s s , rupture or ch an ges in the g ly co ca lix .
R E S U M O
P a r a e s t u d a r a t r a n s f o r m a ç ã o d a c e r c á r i a e m e s q u i s t o s s ô m u l o in vivo, s o b a i n f l u ê n c i a d e u m c o m p o s t o a t i v o c o n t r a o S ch isto so m a m ansoni, f o i e s c o l h i d a a t é c n i c a d a i n o c u l a ç ã o d e c e r c á r i a s n a c a v i d a d e p e r i t o n e a l d e c a m u n d o n g o s . E s t e p r o c e d i m e n t o s e m o s t r o u r e p r o d u z i v e l e p r o m o v e u u m a r á p i d a e f á c i l r e c u p e r a ç ã o d a s l a r v a s a p ó s a l a v a g e m d a s v í s c e r a s a b d o m i n a i s c o m s o l u ç ã o a p r o p r i a d a . O s r e s u l t a d o s o b t i d o s s u g e r e m q u e a l t a s d o s e s d e o x a m n i q u i n e ( v i a i n t r a m u s c u l a r ) , u m a h o r a a n t e s d a i n f e c ç ã o , p r o d u z e m u m e v i d e n t e a t r a s o n a c i n é t i c a d o p r o c e s s o d e t r a n s f o r m a ç ã o d a c e r c á r i a e m e s q u i s t o s s ô m u l o . C e r c á r i a s , c o r p o s c e r c a r i a n o s e e s -q u i s t o s s ô m u l o s f o r a m r e c u p e r a d o s d a c a v i d a d e p e r i t o n e a l d e c a m u n d o n g o s t r a t a d o s e m n ú m e r o s d i f e r e n t e s d a q u e l e s r e c u p e r a d o s d o s a n i m a i s c o n t r o l e s .
P a l a v r a s c h a v e s : S ch isto so m a m a n so n i C e r c á r i a . E s q u i s t o s s ô m u l o . T r a n s f o r m a ç ã o in vivo. O x a m n i q u i n e .
R E F E R E N C E S
1. H o w e lls R E , R a m a lh o - P in to F J , G a z z in e lli G , O liv e ir a C C , F ig u e ir e d o E A , P e lle g r in o J . S c h i s t o s o m a m a n s o n i : th e m e c h a n is m o f c e r c a r ia l ta il lo s s a n d its s ig n if ic a n c e to h o s t p e n e tr a tio n . E x p e r im e n ta l P a r a s ito - lo g y 3 6 : 3 7 3 - 3 7 5 , 1 9 7 4 .
Melo AL, Pereira LH. Kinetics of the cercaria-schistosomulum transformation
in v ivo : 2.The effectofoxamniquine. Revista da
Sociedade Brasileira de Medicina Tropical 18: 251-255, Out-Dez, 1985
3. M e lo A L , P e r e i r a L H . I n h ib ito r y e ffe c t o f o x a m n iq u in e o n th e d e ta c h m e n t o f th e ta il o f S c h i s t o s o m a m a n s o n i . T h e J o u r n a l o f P a r a s ito lo g y 6 6 : 1 0 6 7 - 1 0 6 8 , 1 9 8 0 . 4. M e lo A L , P e r e i r a L H . K in e tic s o f th e c e r c a r ia - s c h is to
so m u lu m t r a n s f o r m a tio n i n v i v o . R e v is ta d a S o c ie d a d e B ra s ile ira d e M e d ic in a T r o p ic a l 18: 1 7 - 2 1 , 1 9 8 5 . 5. O liv e ir a M A , P e lle g r in o J , P e r e i r a L H , V a la d a r e s T E .
M ig ra tio n a n d f a te o f S . m a n s o n i in m ic e t r e a te d w ith o x a m n iq u in e . R e v is ta d o I n s titu to d e M e d ic in a T r o p ic a l d e S ã o P a u lo 18: 2 9 8 - 3 0 0 , 1 9 7 6 .
6. P e lle g r in o J , M a c e d o , D G . A sim p lifie d m e th o d f o r th e c o n c e n tr a tío n o f c e r c a r ie . T h e J o u r n a l o f P a r a s ito lo g y 4 1 : 3 2 9 - 3 3 0 , 1 9 5 5 .
7. P e lle g rin o J , P e r e i r a L H , M e llo R T , K a tz N . A c tiv ity o f s o m e te tr a h y d r o - a n d p y r a z in o q u in o lin e s a g a in s t e a rly d e v e lo p in g fo rm s o f S . m a n s o n i . T h e J o u r n a l o f P a r a s ito lo g y 6 0 : 7 2 3 - 7 2 5 , 1 9 7 4 .
8. P e lle g r in o J , P e r e i r a L H , M e llo R T . P r e lim in a r y la b o - r a to r y tr ia ls o f o x a m n iq u in e a s a p r o p h y la c tic a g e n t in
s c h is to s o m ia s is . R e v is ta d o I n s titu to d e M e d ic in a T r o p ic a l d e S ã o P a u l o 18: 9 7 - 1 0 1 , 1 9 7 6 .
9 . P e lle g r in o J , M e llo R T , P e r e i r a L H . F u r th e r stu d ie s o n th e c h e m o p r o p h y la c tic a c tiv ity o f p y ra z in o q u in o lin e s in e x p e r im e n ta l s c h is to s o m ia s is m a n s o n i. R e v is ta d o I n s titu to d e M e d ic in a T r o p ic a l d e S ã o P a u lo 18: 1 4 9 - 1 5 1 , 1 9 7 6 .
10. P e r e ir a L H , P e lle g r in o J , V a la d a re s T E , M e llo R T , C o e lh o P M Z . A n e w a p p r o a c h f o r s c r e e n in g p r o p h y la c tic a g e n ts in s c h is to s o m ia s is . R e v is ta d o In s titu to d e M e d ic in a T r o p ic a l d e S ã o P a u lo 16: 1 2 3 -1 2 6 , 1 9 7 4 . 11. P e r e i r a L H , P e lle g r in o J , M e llo R T . A c tiv ity o f k n o w n
s c h is to s o m a l a g e n ts o n e a r ly d e v e lo p in g fo rm s o f S. m a n s o n i . T h e J o u r n a l o f P a r a s ito lo g y 6 1 : 2 4 9 -2 5 2 , 1 9 7 5 .
