Gazzinelli, G.

Michalick, M. S. M.

Pellegrino, J.

English

Cadernos Espinosanos (E-Journal)

Rev. Inst. Med. trop. São Paulo 21(3): 115-118, maio-junho, 1979 UDC 616.995.122 CULTIVATION OF SCHISTOSOMA MANSONI CERCARIAL BODIES TO ADULT WORMS M. S. M. MICHALICK (1,), G. GAZZINELLI (2) and J. PELLEGRINO (3) SUMMARY Cercaria! bodies of Schistosoma mansoni develop to adult forms in vitro at the sarne rate reported for schistosomules. When compared with worms obtained from mice the initial rate of development of both is the sarne but it becomes slower at !ater stages. INTRODUCTION The cultivation of S. mansoni cercaria! bodies has been previously reported (COLLEY & WIKEL 3; TIBA et al. 7). In this paper, the _pattern of dievelopment of c·ercarial bodies to adult worms in vitro is described. 'MATERIALS AND METHODS Cercaria! bodies were prepared as follows (RAMALHO-PINTO et al. 6) : 10 ml-aliquots of concentrated intact cercariae were pipetted into 15-ml glass conical centrifuge tubes anel cooled in an ice bath for 10 min to reduce motility of the organism. Following centri-fugation at Iow speed for 1 min the superna-tant fluid was decanted. The pack•ed cerca-riae (10,000-20,000) were suspended in 2 ml of cold sterile Ear1e's balanced saline containing O. 5% lactalbumin hydrolysate, 200 units/ml of penicillin, 200 µg/ml of streptomycin (Elac) and whorled for 45-60 sec in a Vortex Jr. mi-xer (Scientific Industries Inc., Queens Village, N. Y. ) . Isolation of cercaria! bodies. from tails was accomplished by transf.erring a 2. 0-ml sus-pension to another glass conical C'entrifuge tube containing 8.0 ml of Elac; after 3 min at room temperature the tail-rich superríatant was decanted off and the sedimented bodies weve washed 3 times Elac. The bodies thus obtained were transferred to a culture medium (200 organisms/ml) consisting of Elac plus 50% heat-inactivated rabbit serum, and 1 % rabbit red cells (CLEGG 2) and maintained in this medium in 20-ml screw-top tube at 37°C in an atmospher,e of 5% C0,-95.% air. The medium was replaced every 3-4 days when the parasites were observed and counted in an inverted microscope at 100 x magnification. Morphological criteria as described by BARBOSA et al. 1 were used to classify the parasites in relation to tlieir stage. For tech-nical facilities the gastric caecum shape was chosen according to FAUST et al. 4: stage 1 -schistosomula presenting only a light stain which stands for the beginning of the caecum; stagie 2 - a darker stain now bifurcating but not bypassing the acetabulum; stage 3 - the dark stain bypasses the acetabulum and its branches link themselves !ater on: stage 4 -the dark bifurcated stain after reconnection grows to the parasite end, but not longer than the bifurcated -~aecum; stage 5 - the final Iinked caecum grows longer tha;n its bifurca-tJed section, but shorter than three times. of it;& Ienght; stage 6 - their linked caecum grpws three times longer than the bifurcated caiecum (mature adults). Proteolytic activity and protein !l.SSays were performed in the ex-tracts as previously described (GAZZINELLI et al.5). (1) Departamento de ·Parasitologia, Instituto de Ciências Biológicas, UFMG (2) Departamento de Bioquímica-Imunologia, Instituto de Ciúncias Biológicas, UFMG. C. Postal 2486, 30.000 Belo Horizonte, Bbsil (3) Deceased Febrúary, 1977 This work wás supported by CNPq, SIP 08-099 115 MICHALICK, M. S. M.; GAZZINELLI, G. & PELLEGRINO, J. - Cultivation of Schistosoma mansoni cercarial bodies to adult worms. Rev. Inst. Med. trop. São Paulo 21:115-118, 1979. RESULTS First of all an attempt to increase the re-covery of the body was made: mixed cerca-rial bodi,es and tails were sedimented for a lon-ger period of time and this procedure repea-ted 2-3 times. As can be seen in Table I this manipulation increased the death rate in the culture medium. By the standard procedure as described in Materiais and Methods the bodies presen1Jed a mortality rate of 3.44%/ day during the first 10 days which fell to O. 81 %/day cm subsequent days (Fig. 1). Under these conditions more than 90% parasites be-100 100 o .-,:-1--> 50 ~ " ., o t >-1--:::; 50 o <[ 20 1--o: o ::; o 10 15 come water sensitive in about 2 hr. However, the elimination of the preacetabular glands. as me,asured by the residual proteol:ytic activity was completed only about four days !ater (Fig. 1 insert). This did not seem to interfere with the development rate of the parasite• for as shown in Table II the initial rate of develop-ment was the sarne in vitro and in the white mouse, when identical criteria were used to classify the stages of the parasites.. Addition of O. 6% of linolenic, or linoleic acid to the culture media did not stimulate the glandular secretion. 20 25 30 35 TIME (days) Cercaria! Three Ftg. 1 - Mortality rate o'f cercarial bodies in culture media. The points represent the mean of ten tubes containing about 200 cercarial bodies each at day zero. Insert: remnant proteolytic activity in cercarial bodies cul-tured under the sarne conditlons,,. TABLE I Death rate of cercaria! bodies during the first four days in culture media Percent mortality on day bodies preparations (*) 1 2 3 min sedimentation 3.0 4.1 4.9 (1 time) Ten min sedimentation 2.0 7.8 18.2 (3 times) 4 7.1 26.0 (*) Cercaria! suspensions were agitated in a vortex mixer and the bodies separated from the tai! by gravity se-dimentation 116 MICHALICK, M. S. M.; GAZZINELLI, G. & PELLEGRINO, J. - Cultivation of'Schistosoma mansoni cercaria! bodies to adult worms. Rev. Inst. Med. trop. São Paulo 21:115-118, 1979. ln vitro development as compared to in vivo took plaoe at the sarne rate up to the stag.e 2 and then slowed down so that only 33 % parasites reached the stage 4 after 34 days in the culture. At this time 69% of the parasites have already attained maturity (sta-ge 6) in the mouse (Table II). This pattern of development was highly reproducible if conditions such as pH and number of bodies per tube were maintained strictly constant. After 50 days of experiment half of the para-sites were still alive although assynchronic growth and development were always obser0 ved. TABLEII Development of S. mansoni cercaria! bodies in vitro compared with optimum development in the white mouse PERCENT OF LIVING PARASITES Stages 1 2 3 4 5 6 Days in in in in in in in in in in in in vivo(*) vitro vivo vitro vivo vitro vivo vitro vivo vitl'O vivo vitro 7 100 2 o 98 o o o o o o o o 9 67 o 33 100 o o o o o o o o 13 14 o 32 82 50 18 4 o o o o o 22 5 o 9 31 19 51 56 18 11 o o o 34 o o 1 19 3 46 6 33 30 2 60 o 46 o o 3 o 1 o 3 o 13 o 80 o 49 o o o 4 o 36 o 40 o 20 o o (*) Data obtained from BARBOSA et al. 1 for the sarne strain of parasites used in this work (L.E. strain, Belo Horizonte) DISCUSSION Schistosoma mansoni can be cultured in vitro from cercaria! bodies to a stage of de-vielopment at which worms are nearly sexual-. ly mature. The prepamtion of the bodies is. very simp1e and they can be obtained in a large amount. Cercari:al bodies developed in vitro at the sarne rate and to sarne stage as those reported by CLEGG 2 for schistosomula. A deviation from the optimum growth rate obseryed for schistosomules in the mouse has~ also been noted when dealing with cercada! bodtes ('I1able II). Since the preparation of cercaria! bodies is simpler than of schistoso-mula it might be adventageous to use cerca-ri:al bodies for culture studies. RESUMO Desenvolvimento em cultura de corpos cercarianos do Schistosoma mansoni Estudou-se o desenvolvimento de corpos cercarianos in vitro. O comprimento do ceco gástrico, de fácil observação, foi utilizado co-mo critério para medida do desenvolvimento. Constatou-se que rapidamente os corpos cer-carianos se transformam em esquistossômulos a julgar pelo aparecimento da sensibilidade a soluções hipotônicas e pela eliminação da •ati-vidade proteolítica das. glândulas cefálicas. Verificou-se, ainda, que o desenvolvimento dos corpos cercarianos são assincrônicos, seguindo o mesmo curso de desenvolvimento do esquis-tossômulo cultivado in vitro. Por outro lado, ambos, corpos cercarianos e esquistossômulos, sofrem um desvio do crescimento ótimo quan-do comparados com o parasita desenvolvido no camundongo. ACKNOWLEDGEMENTS We thank José de Souza Filho and Rita de Cassia Soares for technical assistance. REFERENCES 1. BARBOSA, M. A.; PELLEGRINO. J.; COELHO, P. M. Z. & MACHADO, I. B. M. - Quantita-tive aspects of the migration and evolutive assyn-chronism of Schistosoma mansoni in mice. Rev. Inst. Med. trop. São Paulo 20: 121-182, 1978. 2. GLEGG, J. A. - ln vitro cultivation of Schisto-soma mansoni. Exptl. Pa.rasit. 16: 133-147, 1965. 3. COLLEY, D. G. & WIKEL, S. K. - Schistoso-ma mansoni: simplified method for the prodnc-ºil7 MICHALICK, M. S. M.; GAZZINELLI, G. & PELLEGRINO, J.· - Cultivation of Schistosoma mansoni cercaria! bodies to adult worms., Rev. Inst. lUetl. trolJ. São Paulo 21:115-118., 1979. tion of schistosomules. Ex1Jtl. Parasit. 35: 44-51, 1974. 4. FAUST, E. C.; JONES, C. A. & HOFFMAN, W. A. - Studies on schistosomiasis mansoni in Puer-to Rico. III - Biological studies. 2. The mam-malian phase of the Iife cycle. Puerto Rico J. Publ. Health & Trop. lUetl, 10: 133, 1934. 5. GAZZINELLI, G.; OLIVEIRA, e. e.; FIGUEI-REDO, E. A.; PEREIRA, L. H.; COELHO, P. M. Z. & PÊLLEGRINO, J. - Biochemical eviden-ce for morphogenetic change of cercaria to schis-tosomule. Exptl. Parasit. 34: 181-188, 1973. 6. RAMALHO-PINTO, ;F. J.; GAZZINELLI, G.; HO-118 WELLS, R. E.; MOTA-SANTOS, T. A.; FIGUEI-REDO, E. A. & PELLEGRINO, J. - Schistoso-ma mansoni: defined system for stepwise trans-formation of cercaria to schistosomule in -vitro. Exptl. Parasit. 36: 360-372, 1974. 7. TIBA, Y. ; HOLANDA, J. C. ; RAMALHO-PIN-TO, F. J.; GAZZINELLI, G. & PELLEGRINO, J. - Schistosomula (Schistosoma. mansoni) obtai-ned in vitro: viability in culture and infectivity for mice. Trans. Ro:yal Soe. Trop. l\letl. & Hyg. 68: 72, 1974. Recebido para publicação. em 22/9/1978. 

Desenvolvimento em cultura de corpos cercarianos do Schistosoma mansoni

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Cultivation of schistosoma mansoni cercarial bodies to adult worms

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