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Revista da Sociedade Brasileira de Medicina Tropical 37( 6) :502-504, nov-dez, 2004
The effect of
Zym om on as m obilis
cultur e on exper imental
Schistosom a m an son i
infection
O efeito da cultura de
Zym o m o na s m o bilis
na infecção experimental
por
Schisto so m a m a nso ni
Juliana de Fátima Macedo Santos
1, Joelma Vasconcelos
1, Joelma Rodr igues de Souza
2, Er idan
de Medeir os Coutinho
2, Silvia Mar ia Lucena Montenegr o
2and Eulália Azevedo-Ximenes
1ABSTRACT
C5 7 Bl/1 0 m a le m i c e i n f e c te d wi th Sc histo so mamanso ni we re d i stri b u te d i n to m i x e d , p ro p h yla c ti c a n d c u ra ti ve gro u p s. A c u ltu re o f Zymo mo nasmo b ilis wa s o ra lly a d m i n i ste re d to m i c e . A 6 1 % p ro te c ti o n f ro m th e i n f e c ti o n wa s o b se rve d i n th e c u ra ti ve gro u p ( p < 0 .0 5 ) . Hi sto p a th o lo gi c a l stu d y o f th e li ve rs a n d i n te sti n e s sh o we d si m i la r re su lts.
Ke y-words:Zymo mo nas mo b ilis. Sc histo so ma manso ni. Im m u n o p o te n ti a to rs. Ex p e ri m e n ta l stu d i e s.
RESUMO
Ca m u n d o n go s C5 7 Bl/1 0 d o se x o m a sc u li n o , i n f e c ta d o s c o m Sc histo so ma manso ni f o ra m d i stri b u í d o s n o s gru p o s m i sto , p ro f i lá ti c o e c u ra ti vo . Cu ltu ra d e Zymo mo nas mo b ilis f o i a d m i n i stra d a o ra lm e n te a o s c a m u n d o n go s. Um a p ro te ç ã o d e 61% f o i o b se rva da n o gru po c u ra ti vo ( p< 0,05) . Os e stu do s hi sto pa to ló gi c o s do s f í ga do s e i n te sti n o s m o stra ra m re su lta do s si m i la re s.
Pal avr as-chave s: Zymo mo nas mo b ilis. Sc histo so ma manso ni. Im u n o p o te n c i a d o re s. Estu d o s e x p e ri m e n ta i s.
1 . De par tme nt o f Antib io tic s o f the Fe de r al Unive r sity o f Pe r namb uc o , Re c ife , PE, B r azil. 2 .De par tme nt o f Immuno lo gy, Ce ntr o de Pe sq uisas Agge u Magalhãe s, Fundaç ão Oswaldo Cr uz, Re c ife , PE, B r azil.
Ethic s Co mmitte e in Use o f Animal ( CEUA/FIOCRUZ) n0: P0 0 9 1 -0 1
This wo r k was suppo r te d b y CNPq ( Co nse lho Nac io nal de De se nvo lvime nto Cie ntífic o e Te c no ló gic o , B r azil) .
Addr e ss to: Dr ª Silvia Mo nte ne gr o . De par tme nt o f Immuno lo gy,Ce ntr o de Pe sq uisas Agge u Magalhãe s/CPq AM/Fundaç ão Oswaldo Cr uz. Av. Mo r ae s Re go s/n, Cidade Unive r sitár ia, 5 0 6 7 0 - 4 2 0 Re c ife , PE, B r azil
Fax: 5 5 8 1 3 4 5 3 - 1 9 1 1 - Te l: 5 5 8 1 2 1 0 1 - 2 5 6 5 e - mail: silvia@ c pq am. fio c r uz. b r
Re c e b ido par a pub lic aç ão e m 2 6 /2 /2 0 0 4 Ac e ito e m 2 5 /8 /2 0 0 4
COMUNICAÇÃO/COMMUNICATION
The immune response c an be non-spec ific ally stimulated by a number of agents9 1 2 . One of these, Zym o m o na s m o b ilis, has already been studied6, and its antagonistic effects against bacteria, fungi and protozoa are known6 7.
Although Z. m o b ilis has been used in several biologic al systems7, little is known about its ac tion on helminthic infec tions. Sc histosomiasis was studied on ac c ount of the severity the disease assumes in many c ases and the organic defic ienc y it produc es, making it one of the most serious public health problems.
The present study focused on the evaluation of host protection and the granulomatous lesions around Schisto so m a m a nso ni eggs. Six to eight week old C5 7 Bl/1 0 male mic e were obtained from the animal fac ilities of CPqAM-FIOCRUZ, Rec ife, PE, Brazil. Experimental and c ontrol groups were infec ted perc utaneously
with 1 0 0 c erc ariae ( BH strain) shed from laboratory infec ted Bio m pha la ria gla b ra ta.
A standard 1 09 CFU/mL c ulture of Zym o m o n a s m o b ilis was administered orally, at a dose of 0 .3 mL/day 8.
For evaluation of the resistanc e to the S. m a nso ni infec tion and granuloma formation, the experimental groups were divided into prophylac tic ( n = 1 8 ) and c urative ( n = 1 7 ) in ac c ordanc e with the guidelines for the administration of the Z. m o b ilis culture, for 7 days before or 7 days after the infection, respectively. A mixed group ( n = 1 5 ) , rec eiving the bac terial c ulture before and after S. m a nso ni infec tion was also studied.
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Sa nto s JFM e t al
but ingested the bac terial c ulture. In the plac ebo group ( n = 5 ) , the animals ingested c ulture medium without bac teria and ac ted as c ontrols for the c ontrol 2 group.
The degree of protec tion was measured by infec ting the mic e and perfusion of the liver 6 0 days later. The number of worms rec overed from eac h treated group of mic e was c ompared with the non-treated c ontrol group, and protec tion was expressed as a perc entage ac c ording to the following formula:
Pr o te c tio n = C-E/C x 1 0 0 , whe r e :
C – is the numb e r o f wo r ms in the c o ntr o l gr o up;
E – is the numb e r o f wo r ms in the tr e ate d gr o up.
Mic e fro m the experimental and c o ntro l 1 gro ups were sacrificed on the 6 0 th day of infection and samples from the liver and intestines were fixed in 1 0 % buffered formalin, embedded in paraffin, sectioned at 5
µ
m and stained with hematoxylin-eosin.Statistic al c omparisons were made using Student’s t test, with a level of signific anc e for the p value set at 0 .0 5 . All the experiments were repeated three times.
The total number of adult worms rec overed was signific antly lower in the c urative group as c ompared to the c ontrol 1 group ( p = 0 .0 0 0 4 9 ) , resulting in 6 1 % protec tion ( Table 1 ) . In this group the immune response elic ited by Z. m o b ilis oc c urred after the S.m a nso ni infec tion. As the ac tivation of the immune system begins on the third day, tends to peak on the fifth and dec rease on the eighth day1 1, the immune response was very effic ient, with death of the par asites o c c ur r ing at the peak phase. The degr ee
decrease in the number of adult worms was to be expected, since the onset of the curative treatment would provoke a secondary immune response. However, in this group, whic h involved a combination of the two treatments ( prophylactic and curative) , an exacerbation of the schistosomiasis infection was observed, since a larger number of adult worms was collected in comparison with the control 1 ( infected and non-treated) group.
Studies with probiotic bac teria demonstrated that the dose and frequenc y of administration of these preparations influenc e the c ourse of the stimulation of the immune system5. In this study,
Z. m o b ilis c ulture dose administrated daily to the mixed group was similar to that given to the prophylac tic and c urative groups, but there was an inc rease in the frequenc y of administration, from 7 to 1 4 days. The observed inc rease in frequenc y may have led to an anergy state in this group, allowing a greater number of adult worms to be rec overed at the time of the perfusion.
As far as histopathology was c onc erned, the lesions were similar in the experimental and control 1 groups. Hepatic changes were c harac terized by numerous periovular granulomas, mainly of the exudative type. Eggs and granulomas were seen in all the histologic al layers, in the three intestinal segments, though were more abundant in the walls of the jejunum-ileum.
No statistic ally signific ant diffe r e nc e was o b se r ve d in the we ights o f the sple e n and live r o f the mic e fr o m the gr o ups infe c te d with S. m a n so n i. The same was tr ue fo r the gr o ups th a t we r e n o t in fe c te d ( Ta b le 2 ) . Th e la tte r r e s ults in c o m b ina tio n with the histo pa tho lo gic a l a na lysis o f the se o r ga n s in th e c o n tr o l 2 ( tr e a te d a n d n o n - in fe c te d) a n d plac eb o ( no n-tr eated and no n-infec ted) gr o ups, c o r r o b o r ate the c o nc lusio n r e ac he d b y Fo x4 that pr o b io tic s administe r e d o r ally do no t po se a thr e at o f damage to the live r and sple e n.
Table 1- Protection against Schistosoma mansoni infection in m ice subm itted to different thera peutic schem es with cultures o f Zymomonas mobilis.
Groups Adult worm recoverya
Protection %
Prophylactic ( n = 1 8 ) 1 3 .7 2 ± 7 .2 5 24
Curative ( n = 1 7 ) 7 .0 0 ± 6 .6 6 * 61
Control 1 ( infected/non treated) ( n = 1 7 ) 1 8 .0 0 ± 9 .6 0
a
Mean ± standard deviation, n = number of mice
* Statistically significant in comparison to control 1 group ( p = 0 .0 0 0 4 9 0 )
o f p r o te c tio n r e p o r te d is c o m p a r a b le to th e im m un ity c o n fe r r e d b y s o m e s c h is to s o m ia s is va c c in e c a n dida te s3. Pr e vio us studie s c ar r ie d o ut with the immuno po te ntiato r s Co ryn e b a c te ri u m pa rvu m1 1 3 and levamiso le1 0 using a similar me tho do lo gy, de mo nstr ate d that the e ffe c ts o n the c ur ative gr o up tr e ate d with le vamiso le we r e similar to tho se o b taine d with the c ultur e o f Z. m o b i li s and diffe r e nt fr o m the r e sults with the use o f C. p a rvu m. The var iatio n in the e ffe c ts o f immuno po te ntiato r s de pe nd o n se ve r al fac to r s suc h as: ho st immune status, se ve r ity o f infe c tio n, do se , timing o f dr ug administr atio n and spe c ific ac tio n o f e ac h o ne1 1 3.
The r e fo r e , the pr o phylac tic gr o up was no t signific antly diffe r e nt whe n c o mpar e d to the c o ntr o l 1 gr o up, sho wing 2 4 % protection against S. m a nso ni infection. In this group, which was treated before the infection, the immune response seemed to be much less efficient. On the other hand, in the mixed group a
Ta ble 2 - Avera ge liver a nd spleen weights in the experim enta l a nd co ntro l gro ups.
Groups Spleen weighta
( g) Liver weighta
( g)
M ± SD M ± SD
Prophylactic ( n = 1 8 ) 0 .2 5 2 ± 0 .0 5 1 .6 1 ± 0 .2 9
Curative ( n = 1 7 ) 0 .2 4 0 ± 0 .0 3 8 1 .6 2 ± 0 .2 1
Mixed ( n = 1 5 ) 0 .2 5 0 ± 0 .0 6 1 .4 7 ± 0 .2 5
Control 1 ( n = 1 7 ) 0 .2 5 0 ± 0 .0 6 1 .5 0 ± 0 .2 9
( infected/non treated)
Control 2 ( n = 6 ) 0 .1 0 5 ± 0 .0 1 7 1 .2 5 ± 0 .1 6
( non infected/treated)
Placebo ( n = 5 ) 0 .0 9 6 ± 0 .0 2 0 1 .2 4 ± 0 .0 1
( non infected/non treated)
a Mean ± standard deviation
ACKNOWLEDGEMENTS
We are grateful to Luiz Carlo s Figueiredo and Ro ni Evênc io fo r te c hnic al suppo r t.
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