Эта статья опубликована в сборнике по материалы первой Международной юбилейной конференции «Актуальные проблемы инфектологии и паразитологии», посвященной 110-летию со дня открытия проф. К.Н.Виноградовым сибирской двуустки у человека (2-5 апреля 2001, г. Томск)

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Влияние гиперинвазии мирацидиями Schistosoma mansoni на выживаемость улиток Biomphalaria glabrata (Say, 1818).

INTRODUCTION

The planorbid snails of genus Biomphalaria are the intermediate hosts of Schistosoma mansoni. They are involucred in the transformation, miracidia-sporocysts-cercariogenesis. However, there  are restrictions: biochemical, ecological and immulogical that fall into parasite-mollusk interaction. ^5,7,8,10,11 One of the most important aspects on the researching efforts are the complex or contradictory results, like the phenomenon of non-susceptibility which  present some natural populations and /or their descending , snails  born in the laboratory  at  the  exposed experimentally  of miracidia of S. mansoni.^3,5,16,17,18  Diverse strategies   have described which  operate like defense in the intermediate host these include:  phagocytes by the  haemocytes, cytotoxicity, adherence and destroyed sporocysts. ^2,9,15 The purposes of this research  was to explore the influence of the hyperinfection of miracidia of S. mansoni (SM, Venezuela) on the refractory snails survival Biomphalaria glabrata BH (Belo Horizonte, Brazil). Whole obtained results were in order to seek new evidences about the  nature of their relationship between schistosome and non susceptible snails.      

 
MATERIALS AND METHODS

Groups of Biomphalaria glabrata snails of   5-10 mm of diameter were exposed with  miracidia of Schistosoma mansoni,  which maintained on NMRI mice. The feces was clarified by  washing procedures using isotonic saline solution, helping the  capture of miracidia¹³ . The eclosion of the eggs was estimulated during 30 minutes  by exposing them to artificial light fountain. Experimental conditions about the infection, evaluation, maintenance of the parasited-snails  and  controls-snails meet detail described in anterior researches.^12,13 Survival reports  were carried  out weekly, for both controls  and parasitized molluscs.
In experiments A, 20 B. glabrata of Brazilian origin (BH, Belo Horizonte) were exposed to five, ten and fifteen miracidia of S. mansoni of Venezuela origin (SM, Carabobo) independent in duplicated.
In experiments B (preliminary), 20 B. glabrata of Brazilian origin (BH, Belo Horizonte) were exposed to five miracidia of  S. mansoni  of  Venezuela  origin (SM, Carabobo), which were sacrificed when the fourth week arrived for researching sporocysts presence.
In experiments C, 20 B. glabrata of  Venezuela origin (Barbula, Carabobo) were exposed to five miracidia of S. mansoni of Venezuela origin (SM, Carabobo) in duplicated. To the end of this, to seek the evidences in defending the hypothesis of similar patroness of maximum mortality  of the BH refractory snails  (parasited  with  isolated SM) at the prepatent period in associations of high compatibility. Which were described before as very susceptible in sympatric association. ^4,12,13  
In experiments D,  20 B. glabrata  of Brazilian origin (BH, Belo Horizonte) were exposed to five miracidia of S. mansoni of Brazilian origin (BH, Belo Horizonte) independent in duplicated (positive control).
In experiments A, C and D were evaluated  the cercariae, and  the results were expressed like percentages of  positive snails in the time, to determine the  prepatent period.

RESULTS

The survival curves of  B. glabrata (BH)  exposed with  5, 10 and 15 miracidia of  S. mansoni (SM) are represented in  figure 1 (experiment A) respectively.   Those that exposed to  ten and fifteen miracidia, survived until 12-13^th week. A  negative influence between the number of miracidia exposed and survival curves of snails-non susceptible was observed. The snails exposed to  five miracidia  survive up to  20-23 th. week. In  contrast to  those were exposed to ten and fifteen miracidia which survived until 12-13 week respectively.
Moreover,  the  total mortality of the host-snails with 10 and 15 miracidia are concentrate between 4-14th week post-infection. In relation  to molluscs exposed with five miracidia  50%  of them were reduced  during the forth and tenth week.
The figure  2   represents  the  percentage of  B. glabrata positive evaluated  since the fourth week of  infection (experiment C). The observed tendency is a high positively of the snails Barbula which exposed to  five miracidia of the isolate SM. It observed that the molluscs begin to eliminate cercariae since the fourth till the thirteenth week in beginning parasitism. The infection dynamic presented a lapse of ten weeks, in  comparison with the planorbs, survival curve BH opposite  to the SM in the infection with five miracidia is observed (prepatent period). The molluscs population died in 55%. In  figure 3 is represented the percentage of B. glabrata (BH) exposed with five miracidia (BH).
In  figure 4 is represented the  survival curves of  B. glabrata  exposed with  5 miracidia of  S. mansoni,  in combination  Barbula vs. SM and BH vs. BH.

DISCUSSION

It has been described the effect of the hyperinfection of miracidia of digenic trematode on the mortality of snails-host.   Jourdane in  19836  point out the effect of the mortality of  Biomphalaria pfeifferi infected with 10 and 20 miracidia  of Echinostoma togonensis. He founds, that the  number increase of  the infective miracidia  influences on the molluscs mortality. This author reported  that the infected populations  with 10 and  20 miracidia, the molluscs reduced their survival in a  50% among the  6-7^th and 5-6^th week respectively.  These results demonstrated that effectively, increases of the infectives miracidia population play an important roll in the infectious dynamic. The effect of hyperinfection is interpreted like a phenomenon of acceleration in the course of prepatent period, assuming that, in the phase where occurred the maximum mortalities of  snails. In other digenea trematodes, the associations have presented equal behavior. Lymnae gedrosiana infected by  Ornithobilharzia turkestanicum and Oncomelania hupensis exposed from Schistosoma japonicum .^6,10
The results pointed out in this research, let  infer that the studied  molluscs are refractories because no cercariae are produced and high mortality rate is observed in  Figure 1; the dynamic  of mortality  behaves as if is a good association. This  phenomena is explained at  the biochemical, physiological and morphological levels of intermediary host that produce  death of snails.^1,13,14 The  found  results defend the evidences that the prepatent period of parasitcs are surpose to the intermediary-host mortality inclusive in low compatibility associations. This phenomenon is evidenced in the parallel experiments did in the Barbula infections opposite SM, in which the elimination of cercariae (4-13 week) coincide  with the maximum snails mortalities of  BH (Figure 2, experiment C) and  in experiment D (positive control) in which elimination of cercariae is observed  during octave week to post-infection (Figure 3, experiment D).
We express our sincere thanks to Mr. Ysrael Mбrquez is who professor at the Simуn Rodrнguez University, because of  his English translation of the manuscript.    The technical assistance of  Beatriz Moy, Senecio Rivas, Claudia Roman, Josй Castellanos and Jorge Lopez is gratefully achnowledgments.
In the same association, but with parasitical loads of 10 and 15 miracidia, in that same period of time the mortality is almost total. Which indicate that in spite of that  the snails BH didn’t get eliminate cercariae, the development of atrophied sporocysts occurred. This fact was confirmed when  snails-parasitic (BH vs. SM) and aften of  four weeks  they were  detected atrophied aspect.

REFERENCES

1. Bayne,  C. and E. Loker, 1987. Survival within  the snail host, P. 321-346.  In:  D. Rollinson and A.  Simpson  (ed.). The  Biology of Schistosomes from genes  to letrines, Academic Press, London.
2. Boehmler, A.M.; Fryer, S.E. and Bayne, C. 1996. Killing of Schistosoma mansoni sporocysts by Biomphalaria glabrata hemolymph  in vitro: Alteration of hemocyte behavior after poly-L-lysine treatment of plastic, and  kicetics of killing by different host strains J.  Parasitol. 82 :332-335
3. Dias, G .;  J. Bruce and  G. Coles. 1988. Strain variation in  the infectivity  of Schistosoma  mansoni from Biomphalaria glabrata.  Rev. Inst. Med. Trop. Sao  Pablo.  30 : 86-90.
4. Incani, R.N. 1990.  Heterogeneidad  genetica en Schistosoma  mansoni.  Trabajo de  Ascenso,  Universidad de Carabobo, Venezuela. 129 p.
5. Jones, J.;  C.N. Helm.  and  J.R. Kusel. 1988. Variation in susceptibility of Schistosoma mansoni to  damage  by  polycations.  Mol.  Bioch.  Parasitol.  30:35- 44
6. Jourdane, J. 1983. Effet des  hyperinfestations par Echinostoma togoensis Jourdane et Kulo, 1981  sur  la  croissance et la durйc de  vie du Mollusque  Biomphalaria pfeifferi. Ann. Parasitol. Hum. Comp. 58(2): 103-108
7. Jourdane, J.and  A.Theron,  1987.  Larval developmet; eggs to  cercariae,  p.  83- 113 In : D.  Rollinson  and A. Simpson (ed.).  The  Biology  of Schistosomes  from  genes  to  letrines,  Academic  Press, London.
8. Lie, K.J.; K. Jeong  and D.  Heyneman .  1980.  Tiss  reactions induced by Schistosoma mansoni in B.  glabrata. Ann.  Trop. Med. Parasitol.  74: 57-166
9. Lodes,  M.J. ; Connors, V.A.  and Yoshino, T. 1991. Isolation and fuctional characterization of snail  hemocyte-modulating polypeptide from primary sporocysts of Schistosoma mansoni.  Molecular and Biochemical. 49: 1-30
10. Massoud,  J. 1974.  The effects  of  variation in miracidial  exposure  dose on laboratory  infections of  Ornithobilharzia  turkestanicum  in  Lymmnae gedrosiana. J. Helminthol. 48:139-144
11. Monû,  H.;  A. Thйron and C.  Combes C. 1986. Interaction  between the Biomphalaria  gabrata Schistosoma mansoni  host-parasite system and  the  non-target molluscs: influence on cercarial  production. J. Parasitol. 72:  10- 416
12. Moreno-Alvarez, M .J.  y  V. Delgado.1993. Susceptibilidad de B. glabrata  de  Venezuela  a aislados de S.  mansoni. IV Congreso de Medicina Tropical. Guayaquil,  Ecuador, Resъmenes pбg. 126.
13. Moreno-Alvarez,  M.J. y V.  Delgado.  1999. Susceptibilidad de Biomphalaria glabrata a Schistosoma mansoni  procedentes de Venezuela y Brasil. Rev.Lat-Amer.  Microbiol. 41:  145-147
14. Niemann,  G. and F. Lewis. 1990. Schistosoma mansoni: Influence of Biomphalaria glabrata wize on susceptibility to infection and resultant cercarial production. Exp. Parasitol. 70 : 286- 292
15. Richards, C.S and  D.J.  Minchella. 1987. Transient non-susceptibility  to Schistosoma  mansoni associated with atrial amoebocytic acculations in the  snails  host  Biomphalaria  glabrata. Parasitol.  95: 499-505
16. Richards,  C.S. and P.C.  Shade,  1987.  The genetic variantion of compatibility in Biomphalaria glabrata and Schistosoma mansoni. J. Parasitol. 73:  1146-  1151
17. Richards,  C.S.;  M.T. Knight. and F.A. Lewis. 1992. Genetics of Biomphalaria glabrata on the outcome  of Schistosoma mansoni  infection. Parasitol. Today,  8: 171-174
18. Romero-Morrel.  C.S. y  G.  Marta.  1978 Estudio sobre la susceptibilidad de Biomphalaria glabrata a la infecciуn con dos cepas de Schistosoma mansoni. Rev. Inst.  Med. Trop.  Sao Pablo.20 : 318-322.

  SURVIVAL CURVES OF SNAILS-NON SUSCEPTIBLE BH EXPOSED SM MIRACIDIA

Figure 1.(Experiment A) 20 B. glabrata  snails-resistent (BH) were exposed to 5 ¦, 10 ♦ and 15 •, miracidia  S.mansoni
(SM) in duplicated.

PERCENTAGE OF SNAILS POSITIVITY

Figure 2. (Experiment C) 20 B. glabrata (Barbula) were exposed 5 miracidia of  S.mansoni (SM)

   PERCENTAGE OF SNAILS POSITIVITY

 Figure 3. (Experiment D) 20 B. glabrata (BH) were exposed 5 miracidia of  S.mansoni (BH)

SURVIVAL CURVES OF SNAILS SUSCEPTIBLE

Figure 4. (Experiment D) 20    B.   glabrata   were  exposed  to 5,  miracidia  S. mansoni
(Barbula vs SM and BH vs BH) in duplicated