Improved culture ofFasciola hepatica in vitro

Smith, Maire A.; Clegg, J. A.

doi:10.1007/bf00941940

Cited by 29 publications

(21 citation statements)

References 9 publications

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“…However, the higher absorption surface of the tegument probably determines its major relevance in drug diffusion from the surrounding medium. This statement is supported by the fact that F. hepatica can survive long periods under in vitro conditions, in the absence of detectable nutrient absorption across the intestine (Smith & Clegg, 1981). Additionally, the higher concentrations of the lipophilic ABZ parent drug recovered in F. hepatica, compared to the more polar sulphoxide metabolite under ex vivo conditions (Alvarez et al 2000) may also contribute to demonstrate the relevance of the trans-tegumental drug passage.…”

Section: Discussionmentioning

confidence: 81%

Comparative assessment of the access of albendazole, fenbendazole and triclabendazole toFasciola hepatica: effect of bile in the incubation medium

2004

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The work reported here describes the comparative ability of albendazole (ABZ), fenbendazole (FBZ) and triclabendazole (TCBZ) to penetrate through the tegument of mature Fasciola hepatica, and the influence of the physicochemical composition of the incubation medium on the drug diffusion process. The data obtained from the trans-tegumental diffusion kinetic studies were complemented with the determination of lipid-to-water partition coefficients (octanol-water) for the benzimidazole (BZD) anthelmintic drugs assayed. Sixteen-week-old F. hepatica were obtained from untreated artificially infected sheep. The flukes were incubated (37 degrees C) over 60 and 90 min in incubation media (pH 7.4) prepared with different proportions of ovine bile and Krebs' Ringer Tris (KRT) buffer (100, 75, 50, 25 and 0% of bile) containing either ABZ, FBZ or TCBZ at a final concentration of 5 nmol/ml. After the incubation time expired, the liver fluke material was chemically processed and analysed by high performance liquid chromatography (HPLC) to measure drug concentrations within the parasite. Additionally, the octanol-water partition coefficients (PC) for each molecule were calculated (as an indicator of drug lipophilicity) using reversed phase HPLC. The 3 BZD molecules were recovered from F. hepatica at both incubation times in all incubation media assayed. The trans-tegumental diffusion of the most lipophilic molecules ABZ and FBZ (higher PC values) tended to be greater than that observed for TCBZ. Interestingly, the uptake of ABZ by the liver flukes was significantly greater than that measured for TCBZ, the most widely used flukicidal BZD compound. This differential uptake pattern may be a relevant issue to be considered to deal with TCBZ-resistant flukes. Drug concentrations measured within the parasite were lower in the incubations containing the highest bile proportions. The highest total availabilities of the 3 compounds were obtained in liver flukes incubated in the absence of bile. Altogether, these findings demonstrated that the entry of the drug into a target parasite may not only depend on a concentration gradient, the lipophilicity of the molecule and absorption surface, but also on the physicochemical composition of the parasite's surrounding environment.

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Section: Discussionmentioning

confidence: 81%

Comparative assessment of the access of albendazole, fenbendazole and triclabendazole toFasciola hepatica: effect of bile in the incubation medium

2004

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“…Similar information on artificial excystment has been previously reported using metacercariae of F. hepatica (Dixon 1966;Smith and Clegg 1981;Fried 1994;Wilson 1998); however, although Paramphistomum and Fasciola belong to the trematode group, there are still problems to be solved to obtain a higher percentage of metacercariae excystment.…”

Section: Discussionmentioning

confidence: 54%

“…This culture medium, combined with human serum, was able to support the survival of some newly excysted F. hepatica up to 14 weeks (Smith and Clegg 1981). Undoubtedly, nutritional requirements for both parasites seem to be different because, at the moment, paramphistomids are not able to survive for such a long period of time.…”

Section: Discussionmentioning

confidence: 98%

“…In a preliminary study, 31 assays for excystment of metacercariae were performed using similar systems reported for Fasciola hepatica (Smith and Clegg 1981;Tielens et al 1981;Wilson 1998), with some modifications. Several changes on their parameters were evaluated such as: time of excystment, time of activation or emergency media, and timing on CO 2 exposition to newly excysted paramphistomids.…”

Section: Artificial Excystment Of Metacercariaementioning

confidence: 99%

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Paramphistomum spp: improved artificial excystment and in vitro culture of immature and adult stages

2007

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The aims of the present study were to establish an adequate excystment method for metacercariae of Paramphistomum spp. and to compare the in vitro survival of paramphistomids in three different culture media. In a preliminary study, 31 different assays were performed solely with the purpose of obtaining the best percentage of metacercariae excystment. Once the best excystment method was selected, 21 further assays using 100 metacercariae/assay were carried out to determine the average percentage of excystment. For the in vitro culture of paramphistomids, the Hedon-Fleig, Rohrbacher, and RPMI-1640 culture media were compared by using 24-well immunoplates containing newly excysted flukes or 50-ml capacity tissue-culture flasks for adult paramphistomids. Results indicated an average of 69.0% excystment, the best culture medium to support the survival of immature and adult stages of the trematode being Hedon-Fleig and Rohrbacher, respectively. The best percentage of survival obtained was on day 4 (80%), with some immature and adult trematodes remaining alive until days 10 and 11, respectively.

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“…In vitro excystation of F. hepatica was performed as previously described [26]. Briefly, metacercariae were incubated for 2 h at 37°C in a CO 2 -saturated sodium dithionite solution (20 mM).…”

Section: In Vitro Excystation Of Juvenile Flukes: Viability Estimationmentioning

confidence: 99%

Early and transient cytotoxic response of peritoneal cells from Fasciola hepatica-infected rats

Sibille¹,

Tliba²,

Boulard³

2004

Vet. Res.

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-Experimental infection by F. hepatica was performed on rats. Early recruitment of the peritoneal cell population was observed and revealed transient parasite-killing activity, preceded and followed by a state of total unresponsiveness. The activation peaked at seven days postinfection (dpi) and was characterised by a massive peritoneal cell recruitment, a strong superoxide anion and nitric oxide (NO) production, that were coincident with the fasciolicide activity of these cells, as monitored by an in vitro decrease of juvenile fluke viability in a conditioned medium. The addition of L-NG-monomethyl arginine (LNMMA) to cell cultures abrogated both fasciolicide activity and NO production. Parasites started to die when NO production exceeded 25 µM and all juvenile flukes were killed by a 90 µM NO exposition (Lethal Dose 50 between 45.8 and 50.3 µM, 95% fiducial limits). However, when rat peritoneal cells were cultured in the presence of either infected or control rat serum, juvenile flukes were much more resistant to the oxidative burst, despite a massive attachment of rat peritoneal cells to the parasite tegument. These data suggest that a transient control of fasciolosis may take place in the peritoneum following the parasite intrusion but that the parasite efficiently scavenges the host cellular response to avoid destruction.

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Improved culture ofFasciola hepatica in vitro

Cited by 29 publications

References 9 publications

Comparative assessment of the access of albendazole, fenbendazole and triclabendazole toFasciola hepatica: effect of bile in the incubation medium

Comparative assessment of the access of albendazole, fenbendazole and triclabendazole toFasciola hepatica: effect of bile in the incubation medium

Paramphistomum spp: improved artificial excystment and in vitro culture of immature and adult stages

Early and transient cytotoxic response of peritoneal cells from Fasciola hepatica-infected rats

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