E
c h in o c o c c u s g r a n u l o s u s:
first r e p o r t o f m ic r o c y s t s f o r m a t io nFROM PROTOSCOLECES OF CATTLE ORIGIN USING THE IN VITRO VESICULAR CULTURE TECHNIQUE
E L IS S O N D O M .C .*,**, D O P C H IZ M .C .** * , B R A SESC O M .* & D E N E G R I G .***
S u m m a ry:
The aim o f this w o rk w a s the achievem ent o f microcysts form ation from protoscoieces o f E. granulosus o f cattle orig in using the in vitro vesicular culture technique. Vesiculated protoscoieces and protoscoieces w ith posterior blad ders a p p e a re d du ring the first w e e k o f in cu bation . After 1 4 days o f culture, a lam inated layer a p p e a re d like a fine m em brane in one o f the extremes o f the protoscoieces. O n d a y 2 0 , some microcysts w ith a com plete lam inated layer w e re observed. By d a y 4 8 , microcysts com pletely d e ve lo p e d could be observed. This is the first study w he re microcysts form ation w a s o b ta in e d using protoscoieces of E. granulosus o f cattle o rig in .
KEY WORDS : Echinococcus granulosus,cystic echinococcosis, protoscoieces, in vitroculture.
R é su m é : Ec h in o c o c c u sg r a n u lo su s : p r e m ie r r a p p o r t d’o b t e n t io n DE MICROKYSTES DE PROTOSCOLEX D’ORIGINE BOVINE PAR LA TECHNIQUE DE CULTURE IN VITRO
L 'o b je ctif d e notre étude é ta it l'ob ten tio n d e microkystes de protoscolex d 'E . granulosus d 'o rig in e bo vine en utilisant la technique d e culture in vitro. Des pro tosco le x vésiculeux et des protoscolex av e c une vésicule postérieure sont ap pa rus durant la p re m ière sem aine d 'in cu b a tio n . A près 14 jours d e culture, la m em brane la m inaire a p p a ra ît com m e une fine m em brane à l'une des extrémités des protoscolex. A utour du jo u r 2 0 , quelques microkystes av e c une m em brane la m inaire com plète sont observés.
A u jo u r 4 8 , des microkystes com plètem ent dé ve lop pé s sont obtenus. C 'e st la prem ière fois que l'o n ra p p o rte l'obtention de microkystes d e pro tosco le x d ’E. granulosus d ’o rig in e bovine.
MOTS CLÉS : Echinococcus granulosus, échinococcose cystique, protoscolex, culturein vitro.
H
ydatid disease is caused by the larval stage of E ch in ococcu s granulosus, which can establish itself in a wide range of intermediate hosts including cattle, sheep, pigs, horses and humans. These hosts become infected by ingestion of eggs in contaminated food or water (Breijo et al., 1998).
Protoscoieces have a dual potential for development;
on one hand, they are the infective stage of the adult parasite in the definitive host, on the other hand, they are capable of reversibly developing into cysts in the intermediate host when released by accidental rupture of the mother cyst (secondary hydatidosis) (Lightow- lers, 1990).
The first attempts of in vitro cultivation of E. granulosus protoscoieces were made by Dévé (1901, 1902).
However these assays were unsuccessful because pro
toscoieces died after 28 days in in vitro conditions.
The first successful in vitro cultivation in a cystic direc-
* Laboratorio de Zoonosis Parasitarias, Facultad de Ciencias Exactas y Naturales, Universidad nacional de Mar del Plata, Argentina.
** Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).
Correspondence: Dr. Guillermo Denegri , Laboratorio de Zoonosis Parasitarias, Departamento de Biología, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3250 (7600) Mar del Plata, Buenos Aires, Argentina.
Tel.: (54) (223) 4753150 - E-mail: gdenegri@mdp.edu.ar
tion was reported by Smyth (1962). Since then, a signi
ficant progress was made using a variety of media and conditions (Benex, 1968; Brudnjak et al., 1970; Heath
& Osborn, 1976; Rogan & Richards, 1986; Casado et al., 1986; Rodriguez-Caabeiro & Casado, 1988; Casado
& Rodriguez-Caabeiro, 1988a & b).
Rodriguez-Caabeiro & Casado (1988) worked with hydatid cysts of sheep origin. They observed micro
cysts formation after 15 days. Ponce Gordo & Cuesta Bandera (1997) worked with cysts of sheep, cattle, horse, pig and human origin. Working with this mate
rial, they found differences not only in the time of microcysts formation, but also in the development process. They obtained microcysts from protoscoieces of sheep, pig, horse and human origin. The develop
ment to microcysts could not be achieved with mate
rial of cattle origin. Their initial development was normal but in no case microcysts finally formed.
The in vitro cultivation of E. gran u losu s protoesco- leces allows to obtain information applicable to the hydatid disease control programmes and in bioche
mical, inmunological and treatment studies (Casado et al., 1986).
The aim of this work was the achievement of micro
cysts formation from protoscoieces of E. granulosus of cattle origin using the in vitro vesicular culture tech
nique.
Parasite, 2004, 11, 415-418 415
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2004114415
ELISSONDO M.C., DOPCHIZ M.C., BRASESCO M. & DENEGRI G.
Fig. 1. - Protoscoleces obtained of lung bovine cysts. The typical shape of protoscoleces is observed, hooks (H) and calcareus corpuscles (CC).
Dead stained protoscolece (white arrow). Methylene blue exclusion test 1:10.000.
Fig. 2. - Evaginated protoscolece.
Fig. 3 . - Vesiculated protoscolece. An evaginated protoescolece showing a posterior bladder is observed (arrow).
Fig. 4. - Vesiculating protoscolece (three days of culture). Note the increment in the size.
Fig. 5. - Microcyst showing laminated layer (20 days o f culture) (arrow).
Fig. 6. - Microcyst completely developed (48 days o f culture).
416
Note de recherche
Parasite, 2004, 11, 415-418Microcystsformationfromprotoscoleces
MATERIALS AND METHODS
H
ydatid cysts from naturally infected bovine lungs and livers were obtained from two abattoirs located in the southeast of Buenos Aires province, Argentina. Protoscoleces were removed from cysts by aseptic techniques and washed several times with phosphate-buffered saline (PBS) pH 7.2. Viability was assessed by the methylene blue exclusion test (Casado et al., 1986) or by muscular movements, morphological per
fectness of the whole body, and motility of flame cells.
Groups of 1,500 viable and free protoscoleces were trans
ferred to Leighton tubes, one to five replicates per sample, depending on quantity of available protoscoleces. The cul
ture medium was 199 (Gibco) containing 100 IU peni
cillin, 100 ug/ml streptomycin, 4 mg/ml glucose and 20 % (v/v) foetal calf serum. Cultures were maintained at 37° C and the medium was changed every three- four days. A total of 10 samples (seven from lung and three from liver) was cultured. Development was followed microscopically every day. Development time until cysts was determined in days. The data recorded for each sample correspond to the moment when a stage was rea
ched by the most advanced individuals of the culture.
RESULTS
A
t the beginning of the culture protoscoleces were invaginated (Fig. 1). After 48-72 hours of incubation some evaginated protoscoleces could be observed (Fig. 2). The amount of them is variable in the different cultures, between 5 and 20 %, but it never reached 100 %.Vesiculated protoscoleces and protoscoleces with pos
terior bladders (Figs 3 & 4) appeared during the first week of incubation. Moreover, a considerable incre
ment in the size of them could be observed.
After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the pro
toscoleces. Seventy percent of all samples reached the microcyst formation (57.1 % of lung and 100 % of liver samples). On day 20, some microcysts with a complete laminated layer were observed (Fig. 5). Some samples showed microcyst formation between 24-38 days. By day 48, microcysts completely developed could be observed (Fig. 6). In all cultures, the total number of microcyst formed was approximately 1.6 % of the number of initial cultured protoscoleces. Some cul
tures could be maintained until day 95.
DISCUSSION
T
his is the first study where microcysts formation was obtained using protoscoleces of E. g ra n u losus of cattle origin.As Smyth described (1962), we found two patterns of cystic development in vitro. In the first, some protos
coleces became swollen or vesicular within a few days of culture and grew into thin walled cysts. In the non-vesicular pattern of development, the protosco
leces each develop a posterior bladder which increased substantially in size; eventually a spherical cyst formed and this became enveloped by a laminated layer. Such cysts were morphologically indistinguishable from cysts that developed from vesicular protoscoleces. This also coincides with the works done by Casado & Rodriguez- Caabeiro (1988a) and Ponce Gordo & Cuesta Bandera (1997).
In this study, the time for microcyst formation in days was determined between 20 and 38. Working with sheep samples, Ponce Gordo & Cuesta Bandera (1997) observed microcyst formation between 19 and 37 days and Casado & Rodriguez-Caabeiro (1988a & b) at day 15.
By the other hand, the results of this work with cattle samples are clearly different from those indicated by Ponce Gordo & Cuesta Bandera (1997). A possible explanation could be the use of a different culture medium. Another reason that could explain the diffe
rence is the possibility of a different genotype. The material used for the in vitro culture was not yet cha
racterized by molecular techniques to determined the involved strains, but only the G1 strain was identified in cattle isolates of Buenos Aires Province (Rosenzvit et al., 1999, 2002; Kamenetzky et al., 2002).
The obtention of microcyst of E. gran u losu s should allow us to test in vitro different new anthelminthic drugs for the human treatment of hydatid disease.
ACKNOWLEDGEMENTS
T
he authors thank Dr. González, Dr. Tessi, Dr.Viglietti and Sr. Chasma for their contributions and Paola Otero and Lidia Formaini for provi
ding linguistic assistance. Partial financial support of this work was provided by Fundación Alberto J. Roem- mers.
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Reçu le 6 avril 2004 Accepté le 30 juillet 2004
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