2 1 8
Revista da Sociedade Br asileir a de Medicina Tr opical 3 7 ( 3 ) :2 1 8 -2 2 1 , mai-jun, 2 0 0 4
Behavior of
Schistosom a m an son i
-induced histopathological lesions in
Biom phalaria glabrata
submitted to ionizing r adiation
Comportamento das lesões histopatológicas induzidas pelo Schisto so m a m a nso ni
em
Bio m pha la ria gla bra ta
após radiação ionizante
Car ine M. Azevedo
1, Claudia Cunha Bor ges
2and Zilton A. Andr ade
3ABSTRACT
Pre se nt re po rt de m o nstra te s tha t re pe a te d ra dia tio n o f Sc histosoma mansoni-infe cte d Biomphalaria glabrata, to ta ling 15,000 ra ds, ca use d a sudde n, a lb e it tra nsie nt, suppre ssio n o f ce rca ria l she dding. Initia lly, spo ro cysts pra ctica lly disa ppe a re d fro m the sna il tissue s. The m o re re sista nt de ve lo ping ce rca ria e pre se nte d nucle a r clum ping a nd va cuo la tio n, b e fo re unde rgo ing lysis. No ho st tissue re a ctio n wa s e vide nt a t a ny tim e . Thirty- fo ur da ys a fte r the la st irra dia tio n, the sna ils re sum e d ce rca ria l e lim ina tio n. By tha t tim e num e ro us spo ro cysts a nd de ve lo ping ce rca ria e we re de te cte d, disse m ina te d thro ugho ut sna il tissue s in a pa tte rn sim ila r to tha t o f a highly m a ligna nt ne o pla sm , with no signs o f ho st ce llula r re a ctio ns, which o n the o the r ha nd we re pre se nt in no n- irra dia te d infe cte d co ntro ls. The re gio n o f the o vo - te stis wa s a ppa re ntly de stro ye d a fte r ra dia tio n, b ut re turne d to its no rm a l a ppe a ra nce a ro und 40 da ys a fte r the la st ra dia tio n. Io nizing ra dia tio n a ffe cte d b o th ho st a nd pa ra site in S. m a nso ni- infe cte d Bio m pha la ria gla b ra ta , b ut the re sulting im pre ssive cha nge s we re so o n re ve rse d.
Ke y-words:Sc histo so ma manso ni.B io mphalar ia glab r ata. Io n i zi n g ra d i a ti o n .
RESUMO
O p re se n te tra b a lh o d e m o n stra q u e a i rra d i a ç ã o re p e ti d a , n u m to ta l d e 1 5 .0 0 0 ra d s, re su lta n u m a rá p i d a su p re ssã o d a e li m i n a ç ã o da s c e rc a ri a s e m c a ra m u jo s i n f e c ta do s pe lo Sc hi sto so m a m a n so n i . In i c i a lm e n te o s e spo ro c i sto s de sa pa re c e m d o s te c i d o s. As f o rm a s e vo lu ti va s d a s c e rc a ri a s sã o m a i s re si ste n te s e a p re se n ta m va c u o li za ç ã o c i to p la sm á ti c a e c o n d e n sa ç ã o n u c le a r a n te s d e d e sa p a re c e re m . Nã o f o ra m o b se rva d a s re a ç õ e s n o s te c i d o s d o h o sp e d e i ro . Tri n ta e q u a tro d i a s a p ó s a ú lti m a i rra d i a ç ã o , o s c a ra m u jo s vo lta m a e li m i n a r c e rc á ri a s. Nu m e ro so s e sp o ro c i sto s e c e rc á ri a s e m d e se n vo lvi m e n to a p a re c e m i n f i ltra n d o d i f u sa m e n te o s te c i d o s à m a n e i ra d e u m a n e o p la si a m a li gn a , se m si n a i s d e o p o si ç ã o d a p a rte d o h o sp e d e i ro , a q u a l e ra vi sí ve l n o s c o n tro le s i n f e c ta d o s e n ã o i rra d i a d o s. A re gi ã o d o o vo - te sti s a p a re c e u d e stru í d a a p ó s a ra d i a ç ã o , m a s re to rn o u à su a a p a rê n c i a n o rm a l e m to rn o d e 4 0 d i a s m a i s ta rd e . A ra d i a ç ã o i o n i za n te a f e ta ta n to o ho spe de i ro c o m o a s f o rm a s e m de se n vo lvi m e n to do pa ra si to , m a s e sta s a lte ra ç õ e s i m pre ssi o n a n te s sã o lo go re ve rsí ve i s.
Pal avr as-chave s: Sc histo so ma manso ni.B io mphalar ia glab r ata. Ra d i a ç ã o i o n i za n te .
1 . Pr o gr ama Instituc io nal de B o lsas de Inic iaç ão Científic a do Co nselho Nac io nal de Pesquisa da Univer sidade Feder al da B ahia, Salvado r, B A. 2 . Co ur se o f Exper imental Patho lo gy, Fio c r uz/UFB A. 3 . He ad, Lab o r ato r y o f Expe r ime ntal Patho lo gy. Ce ntr o de Pe sq uisas Go nç alo Mo niz da Fundaç ão Oswaldo Cr uz, Salvado r, B A, B r asil Suppo r te d b y PAPES III ( FIOCRUZ) .
Addr e ss to: Dr. Zilto n A. Andr ade . Lab o r ato r y o f Expe r ime ntal Patho lo gy/CPq GM/FIOCRUZ. R. Valde mar Falc ão 1 2 1 , B r o tas 4 1 2 9 5 - 0 0 1 Salvado r, B A, B r asil. Re c e b ido par a pub lic aç ão e m 2 5 /1 0 /2 0 0 3
Ac e ito e m 1 3 /4 /2 0 0 4
A pr e vio us study fr o m this Lab o r ato r y had no te d that a
s tr a in o f Bi o m p h a la r i a gla b r a ta, h igh ly s us c e ptib le to
Sc h i sto so m a m a n so n i, we r e s e e n to s he d le s s a nd le s s c e r c ar iae alo ng the time o f infe c tio n3. It was also o b se r ve d
that suc h c hanging patte r n o f c e r c ar ial she dding pr e se nte d a
go o d h is to lo gic a l c o r r e la tio n . An in itia l pic tur e o f n o n -r e ac tio n ( to le -r anc e ) g-r adually tu-r ne d into o ne o f he mo c yte
p r o l i fe r a ti o n , wi th fo r m a ti o n o f fo c a l e n c a p s u l a ti n g
le s io n s a r o un d dis in te gr a tin g s p o r o c ys ts a n d c e r c a r ia e . Th e s e fin din gs s ugge s te d th e e xis te n c e o f a pr im itive fo r m
o f a c q u i r e d i m m u n i t y i n i n ve r t e b r a t e s . I n o r d e r t o
e x p lo r e fur th e r a lo n g th e s e lin e s , a p la n to e r a dic a te a
pr e vio us s c h is to s o m a l s n a il in fe c tio n with ir r a dia tio n wa s i n i ti a te d.
2 1 9
Ionizing radiation of snails has been used for various purposes.
Michelson and Dubois8 applied low-dose radiation ( 6 0 Co) to
Bi o m p h a la ri a gla b ra ta and o b se r ve d an e nhanc e me nt o f resistance to Schisto so m a m a nso ni infection. Other workers have
used irradiated miracidia to infect B. gla b ra ta in an attempt to
inc rease resistanc e against S. m a nso ni, with apparently good results5 6 7 9.When all irradiated miracidia died within snail tissues,
a strong resistance was observed to a further challenge with normal
miracidia.
At first, our attempt at irradiating S. m a nso ni-infected snails
aimed to eradicate the developing parasite in order to observe
whether such a procedure would stimulate the development of an
equivalent of acquired immunity in the invertebrate. This proved
to be impractical due to the unexpected behavior of the larval
forms of S. m a nso ni within the snail tissues following irradiation.
However, the data obtained by a subsequent histologic al study
revealed several points of interest on the differential susceptibility
of the developing forms of the parasite, and also of the host tissues
with their differential degenerative and regenerative c apac ities
toward radiation damage. Thus, the second part of the research was to observe the behavior of the snail tissue changes upon
re-infec tion. Sinc e no similar data were found in the literature
reviewed, the publication of these results appeared to be of interest.
MATERIAL AND METHODS
Laboratory raised adult Bio m pha la ria gla bra ta from Feira de
Santana strain1 were infected with 2 0 S. m a nso ni miracidia ( FS
strain) freshly isolated from eggs extrac ted from the liver and
intestines of experimentally infected mice. When the snails started
eliminating cercariae they were irradiated with cesium 1 3 7 ( IBL
9 3 7 C Irradiator, type H, Cis Bio International, Gif-sur-Yvette, France)
with the objective of eradicating the infection. At first, 2 4 snails were
irradiated with two doses of 4 ,0 0 0 rads, delivered with an interval of
one week. A similar second trial was made with another 3 0 infected
snails. These were submitted to 4 doses of 2 ,0 0 0 rads each at weekly
intervals, with results comparable to those of the previous group.
Then, radiation doses were changed to 4 sessions of 3 ,0 0 0 rads
each ( 2 4 infected snails) at weekly intervals and 3 doses of 5 ,0 0 0
rads each ( 3 6 infected snails) , at two-day intervals. In these last two
groups cercarial elimination stopped and 3 0 to 4 8 days later the
snails were killed for microscopic examination. Although they were
not yet eliminating cercariae, an extensive dissemination of the
multiplying forms of S. m a nso ni in the tissues was observed in these
animals, as detailed below.
Eac h ir r adiate d gr o up was always matc he d with a c o ntr o l
gr o up o f ab o ut 2 0 no n-ir r adiate d and infe c te d snails. The y
r e gular ly e liminate d c e r c ar iae thr o ugho ut the time o f the
e x p e r i m e n t. Ce r c a r i a l e l i m i n a ti o n wa s m o n i to r e d b y
e xaminatio n o f individual snails plac e d into 1 0 0 ml b e ak e r s
c o ntaining 1 0 ml o f de -c hlo r inate d tap wate r. Eac h spe c ime n
was e xpo se d to b r ight light dur ing an ho ur, fo r c e r c ar ial
she dding and c o unting. The snails we r e e xamine d at var io us
days po st-infe c tio n.
For histologic al examination the snails were submitted to
ane sthe sia with me ntho l c r ystals fo r 4 ho ur s b e fo r e b e ing removed from the shells. The entire snail was plac ed in Bouin’s
fluid during 5 hours for fixation and then transferred to 7 0 %
alc o ho l. Further pro c edures inc luded dehydratio n in 1 0 0 %
alc ohol, c learing in xylol and embedding in paraffin. Sec tions were routinely stained with hematoxylin and eosin.
RESULTS
Irradiated infec ted snails bec ame negative fo r c erc arial
elimination one week following the sec ond radiation, regardless
of the doses used, but 3 4 days later they were seen to resume
elimination. The number of eliminated c erc ariae varied from 3 to 1 1 8 im m e dia te ly b e fo r e r a dia tio n , a n d dr o ppe d to 1
( minimum) and 5 9 ( maximum) when examined one week after
the first radiation. Histologic al examination of the infec ted snails,
both c ontrols and irradiated, during the period of c erc arial elimination, revealed the presenc e of numerous sporoc ysts and
several stages of developing c erc ariae loc ated in several organs
and tissues, espec ially in ovo-testis, digestive glands and the
tubular and sec ular portions of the kidney. ( Figures 1 A and B) . The presenc e of hemoc ytes ( ameboc ytes) was a c onstant feature.
The y fo r me d mild to mo de r ate ac c umulatio ns ar o und the
pa r a s itic s tr uc tur e s , s o m e tim e s a s s um in g a c o n c e n tr ic
distr ib utio n ar o und the m and, r ar e ly, diffe r e ntiating into granuloma-like struc tures (e nca psula ting co m ple xe s) .
The snails examined one to two weeks after the end of the
irradiation sessions, at a time when c erc arial elimination had c ompletely stopped, revealed a different histologic al pic ture.
Hemoc ytes were rarely found, usually appearing isolated in only
a few plac es. No disc ernable sporoc ysts were detec ted after a thorough searc h in various mic rosc opic sec tions. Developing
fo rms o f c erc ariae, espec ially the mo re differentiated o nes,
presented fragmentation, c ondensation of nuc lear material and the presenc e of empty vac uoles of different sizes. ( Figur e 1 C
and D) . Clumps of eosinophilic debris, probably representing nec rotic parasites, were present in several plac es. There was a
mild to moderate degree of edema in several tissues. The ova and spermatozoids c ompletely disappeared from the ovo-testis,
which structure assumed a mosaic aspect formed by empty spaces
delimited by slender and wavy membranes. The spac es delimited by membranes sometimes c ontained remnants of c erc ariae.
Whe n the histo lo gic a l e xa m ina tio n wa s pe r fo r m e d in
ir r adiate d snails that r e sume d c e r c ar ial e liminatio n, the r e appeared numerous sporoc ysts throughout the tissues without
any signs of tissue reac tion. Also, many c erc ariae in several stages
of development were present. Within the ovo-testis parasitic multiplic ation and infiltration appeared more evident than the
signs of tissue regeneration. The latter were represented by c lusters of ova and only a few spermatozoids ( Figur e 1 D and E) .
Oth e r s n a il tis s ue s did n o t e xh ib it th e r a dia tio n - r e la te d
degenerative and regenerative features observed in the ovo-testis.
Snails irradiated four times with 3 ,0 0 0 rads every 7 th day or
thr e e time s with 5 ,0 0 0 r ads a t two - da y in te r va ls s to ppe d
2 2 0
e liminating c e r c ar iae . Afte r 3 0 to 4 8 days the y we r e k ille d
and mic r o sc o pic ally e xamine d. A histo lo gic al pic tur e was
o b s e r ve d e n tir e ly s im ila r to th a t o f th e s n a ils th a t we r e e lim in a tin g c e r c a r ia e a fte r a te m p o r a r y p o s t- r a dia tio n
suppr e ssio n, whic h indic ate d that the y we r e ab o ut to r e sume
c e r c ar ial e liminatio n.
Thus, b o th the snails and the multiplying par asitic fo r ms
pr e se nte d a high de gr e e o f r e sistanc e to io nizing r adiatio n.
In a se par ate e xpe r ime nt we sub mitte d 1 2 adult no n-infe c te d
B. gla b ra ta to r a dia tio n a n d k e pt 1 4 a s n o n - ir r a dia te d c o ntr o ls. Afte r 4 8 days, 3 o f the ir r adiate d snails and two o f
the c o ntr o ls had die d, sho wing that ir r adiatio n was inde e d we ll to le r ate d b y no r mal B. gla b ra ta . Mo r tality o f infe c te d
s n a ils wa s h igh , r e ga r dle s s o f r a dia tio n . I t wa s diffic ult to
o b ta in pr e c is e c o m pa r a tive figur e s b e c a us e m a n y s n a ils
we r e s a c r ific e d dur in g th e e xpe r im e n t fo r m ic r o s c o pic
e xaminatio n.
DISCUSSION
The b e havio r o f the de ve lo ping stage s o f S. m a n so n i
within its inte r me diate snail ho st fo llo wing ir r adiatio n was
str ik ingly r e minisc e nt o f that o f a highly malignant ne o plasm
whe n ir r adiate d. At fir st the pr o life r ating c e lls we r e almo st
c o m ple te ly de s tr o ye d, b ut th is wa s s o o n fo llo we d b y a
2 2 1
recrudescence of cellular multiplication, that diffusely invaded the
host tissues. Also in keeping with what is generally known about the pathology of irradiation, the less differentiated and rapidly
multiplying forms, the sporoc ysts, were the most susc eptible
elements. As expected, the ovo-testis was the most susceptible host
tissue to be affected by ionizing radiation4. Its return to normal
after radiation damage occurred at about the same time the invading
parasites were re-emerging. The other snail tissues appeared more
resistant, since they exhibited no evident destructive lesions that
could be attributed to radiation.
The situation of the defense cells, collectively designated as hemocytes, is a more complex matter. Hemocytes are considered
highly resistant to radiation2. The present study revealed an apparent
decrease in both hemocyte number and function as a result of
radiation. In non-irradiated infected snails these cells were easily found, sometimes accumulated in small focal areas, and eventually
forming the so-called enca psula ting co m plexes around destroyed
parasites. Although they did not altogether disappear during and
after radiation, they became fewer and isolated, and did not form
e nca psula ting co m ple xe s around radiation-destroyed cercariae. Also, when parasite multiplication was fully resumed, no evident
histo lo gic al signs o f hemo c yte pro liferatio n were o bserved.
However, it is possible that hemocyte reactivity would require more time to be fully established, and probably would have become
evident had the time of observation been further extended after
the po st-r adiatio n r e sumptio n o f c e r c ar ial e liminatio n. As
demonstrated by the observations of Lemos and Andrade3 the
apparent tolerant behavior of hemoc ytes at initial infec tion,
progressively turned into one of proliferation and differentiation
of granuloma-like structures with time.
REFERENCES
1 . Andr ade ZA, Sadigur sk y M. Um e studo c o mpar ativo das c e pas Fe ir a de Sa n ta n a ( B a h ia ) e Po r to R ic o do Sc h i s to s o m a m a n s o n i n a in fe c ç ã o e x pe r im e n ta l do c a m un do n go . Me m ó r ia s do I n s tituto Os wa ldo Cr uz 8 0 : 3 7 - 4 0 , 1 9 8 5 .
2 . B e ze r r a FS M, Ma c h a d o J AN, S o u za R LM, Ch a ve s MM, Co r r e a R F, Co e l h o PMZ. Effe c t o f ga m m a r a dia tio n o n th e a c tivity o f h e m o c yte s a n d o n th e c o u r s e o f Sc h i s to s o m a m a n s o n i i n fe c ti o n i n r e s i s ta n t
Bi o m p h a la ri a te n a go p h i la s n a ils . Me m ó r ia s do I n s tituto Os wa ldo Cr uz 9 8 : 7 3 - 7 5 , 2 0 0 3 .
3 . Le mo s QT, Andr ade ZA. Se q ue ntial histo lo gic al c hange s in Bi o m p h a la ri a gla b ra ta dur ing the c o ur se o f Sc h i sto so m a m a n so n i infe c tio n. Me mó r ias do Instituto Oswaldo Cr uz 9 6 : 7 1 9 - 7 2 1 , 2 0 0 1 .
4 . Liar d F, Chir ib o ga J , Pe lle gr ino J , Co lo n J I, Silva RM. Effe c t o f r adiatio n o n the r e pr o duc tive po te ntial o f Bi o m p h a la ri a gla b ra ta. Re vista B r asile ir a de Pe sq uisa Mé dic a e B io ló gic a 1 : 1 5 7 - 1 6 2 , 1 9 6 8 .
5 . Li e KJ , He yn e m a n D . S t u d i e s o n r e s i s t a n c e i n s n a i l s . 3 . Ti s s u e r e a c ti o n s to Ec h i n o s t o m a li n d o i e n s e s p o r o c yte s i n s e n s i ti ze d a n d r e s e n s i ti ze d B i o m p h a la r i a gla b r a t a. J o u r n a l o f P a r a s i to l o gy 6 2 : 5 1 - 5 8 , 1 9 7 6 .
6 . Lie KJ , He yne m an D, Ric har ds CS. Bi o m p h a la ri a gla b ra ta. Te m po r ar y r e duc tio n o f s n a il’s n a tur a l r e s is ta n c e to Sc h i s to s o m a m a n s o n i b y ir r adiate d Ec h i n o sto m a p a ra e n si mir ac idia. Expe r ime ntal Par asito lo gy 4 3 : 5 4 - 6 2 , 1 9 7 7 .
7 . Lie KJ , J e o ng KH, He yne man D. Ac q uir e d r e sistanc e in snails. Induc tio n o f r e s i s ta n c e to Sc h i s t o s o m a m a n s o n i i n B i o m p h a l a r i a gl a b r a t a .
Inte r natio nal J o ur nal o f Par asito lo gy 1 3 : 3 0 1 - 3 0 4 , 1 9 8 3 .
8 . Mi c h e l s o n E H, D u b o i s L. R e s i s ta n c e to s c h i s to s o m e i n fe c ti o n i n
B i o m p h a la r i a gla b r a t a i n d u c e d b y ga m m a r a d i a ti o n . J o u r n a l o f Inve r te b r ate Patho lo lo gy 3 8 : 3 9 - 4 4 , 1 9 8 1 .
9 . S u l l i va n J T, R i c h a r d s CS , J o e LK, He yn e m a n D. Ri b e i r o i a m a r i n i
ir r adiate d mir ac idia and induc tio n o f ac quir e d r e sistanc e in Bi o m pha la ri a gla b ra ta . Expe r ime ntal Par asito lo gy 5 3 : 1 7 - 2 5 , 1 9 8 2 .
