Development and validation of a cell culture based assay for in vitro assessment of anticryptosporidial compounds

Najdrowski, M.; Heckeroth, Anja R.; Wackwitz, C.; Gawlowska, Sandra; Mackenstedt, Ute; Kliemt, Damaris; Daugschies, Arwid

doi:10.1007/s00436-006-0437-z

Cited by 24 publications

(17 citation statements)

References 23 publications

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“…In vitro evaluation of monensin (0.144 µM) was in conformity with the results reported by Najdrowski et al (2007), indicating the reliability of the present curcumin efficacy data in this study. About 65% inhibition of invasion and/or adherence to HCT-8 cells in the presence of 200 µM curcumin was recorded.…”

Section: Discussionsupporting

confidence: 89%

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Effects of curcumin on Cryptosporidium parvum in vitro

et al. 2009

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Cryptosporidium parvum is a zoonotic protozoan parasite having peculiarities among the apicomplexa that could be responsible for its resistance to some drugs and disinfectants against coccidia. The awareness of Cryptosporidium as a health problem in man and animal is increasing and potent drugs are urgently needed. Curcumin, a natural polyphenolic compound, has been found to be active against a variety of diseases including anticarcinogenic, antimicrobial, and antiprotozoal effects. We investigated the effects of curcumin on infectivity and development of C. parvum in a recently established in vitro system combining infection of human ileocecal adenocarcinoma cell cultures with quantification of intracellular parasites by quantitative polymerase chain reaction. Curcumin was found to be effective (>95% inhibition of parasite growth) at 50 microM for 24 h when infected cultures were exposed for more than 12 h. Withdrawal of curcumin after 24 h of exposure did not result in a significant resumption of C. parvum growth. The invasion of host cells by sporozoites (infectivity) was found to be inhibited at least 65% in the presence of 200 microM curcumin. No significant reduction of viability of C. parvum oocysts after incubation with curcumin was recorded. Altogether, curcumin showed promising anticryptosporidial effects under in vitro conditions and deserves further exploration.

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Section: Discussionsupporting

confidence: 89%

“…Oocysts were isolated according to the protocol described by Najdrowski et al (2007). Human ileocecal adenocarcinoma (HCT-8) cells were used for in vitro culture of C. parvum.…”

Section: Methodsmentioning

confidence: 99%

Effects of curcumin on Cryptosporidium parvum in vitro

et al. 2009

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“…Current methods for detecting parasite stages after in vitro development comprise cell culture assays employing microscopy (Armson et al 1999;McDonald et al 1990), immunofluorescence microscopy (Najdrowski et al 2007a;Rochelle et al 2002;Slifko et al 1997 and, enzymelinked immunosorbent assays (Woods et al 1995 and, chemiluminescence immunoassays (You et al 1996), colorimetric in situ hybridization (Rochelle et al 2001), polymerase chain reaction (PCR) (Godiwala et al 2006;Najdrowski et al 2007b;Rochelle et al 2002) and quantitative PCR (Cai et al 2005;Di Giovanni and LeChevallier 2005;Godiwala et al 2006;MacDonald et al 2002;Shahiduzzaman et al 2009). Even host cell-free cultivation in vitro has been reported (Hijjawi et al 2004 and2010).…”

Section: Introductionmentioning

confidence: 99%

“…The new assay is based on the previously published indirect fluorescent antibody technique, combined with the foci detection method (Najdrowski et al 2007a;Slifko et al 1997), but was further adapted to fast screening of high sample numbers by serial dilutions in microplate format. The viability of the host cells was measured by an integrated water-soluble tetrazolium salt assay, which enabled rapid discrimination of direct antiparasitic activity of a test substance from cytotoxic effects against the host cells in the very same assay.…”

Section: Introductionmentioning

confidence: 99%

“…Validation of the assay was carried out using substances for which anticryptosporidial or anticoccidial activities have been reported: Halocur™ (containing halofuginone) (McDonald et al 1990;Najdrowski et al 2007a;Villacorta et al 1991), Deccox™ (containing decoquinate) (Navetat and Cantaloube 2003) and monensin (Armson et al 1999;McDonald et al 1990;Najdrowski et al 2007a;Woods et al 1995 and. Furthermore, silymarin and press cake derived from milk thistle (Silybum marianum [L.] Gaertn.)…”

Section: Introductionmentioning

confidence: 99%

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In vitro determination of anticryptosporidial activity of phytogenic extracts and compounds

et al. 2012

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Cryptosporidiosis caused by Cryptosporidium spp. is an important diarrhoeal disease observed in farm animals and humans, especially in young or immunocompromised individuals. A novel cell culture assay for testing extracts and pure compounds against Cryptosporidium parvum in 96-well microplate format was established and evaluated. It is based on previously described indirect fluorescent antibody techniques and was optimised for higher sample throughput. Rapid assessment of minimal inhibitory concentrations (MICs) was done by checking each well microscopically for the presence or absence of parasite stages. As a novelty, parasite development was quantified by enumeration of clusters of secondary infection (CSI), which typically appeared upon infection with a distinct parasite inoculum after a defined incubation time. Host cell (HCT-8) viability was measured by an integrated non-destructive water-soluble tetrazolium salt assay (WST-1), which facilitated discrimination of antiparasitic activity from possible cytotoxic effects of a test compound against the host cells. Host cell viability was regarded unimpaired when cultures had 75% or more viability when compared to control cultures without test substance. In this study, a maximum density of distinguishable CSI was obtained when cultures were infected with 2.5 × 10(3) oocysts and incubated for 48 h. The applicable inoculum has to be optimised for each batch of oocysts and before each experimental series. Parasite development was inhibited completely by monensin at 134 nM and silymarin at 50 mg/mL. These concentrations were non-toxic to the host cells and comparable to literature data. The percentages of parasite inhibition were determined for monensin and a 50% inhibitory concentration (IC(50)) of 36.6 nM (27.4-45.5) and a 90% inhibitory concentration of 65.9 nM (54.8-90.2) were calculated. The introduced assay is economic because relatively low parasite numbers may be used. If MICs are determined, evaluation is fast, as each well is viewed only briefly under the fluorescence microscope for presence or absence of CSI. Furthermore it is highly critical because only full parasite inhibition is assessed. Counting of CSI is more laborious and time-consuming, but it allows calculation of parasite inhibition rates and parameters like the half maximal inhibitory concentration (IC(50)). This assay shall be used to assess anticryptosporidial activities of various plant waste materials and by-products from the food and the pharmaceutical industries in the course of the EU project SAFEWASTES. Comparison with in vivo models should be performed to further corroborate the results. Automated evaluation by flow cytometry might facilitate higher sample throughput and reduce operator bias.

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Inactivation of exogenous endoparasite stages by chemical disinfectants: current state and perspectives

Daugschies¹,

Bangoura²,

Lendner³

2013

Parasitol Res

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Chemical disinfection is common practice and inevitable to achieve sufficient control over parasites particularly in intensive animal housing systems. To identify suitable chemicals, reliable data on antiparasitic efficacy of disinfectants are required. This review summarizes recently published experience with procedures applied to evaluate the viability of a variety of endoparasites following physical or chemical stress. It is concluded that laboratory models used to assess antiparasitic efficacy of e.g. commercial disinfectants should consider the most resistant stages of both helminths and protozoa, i.e. ascarid eggs and coccidia oocysts. To ensure reproducibility and transparency, standardized protocols are pivotal. Such protocols are established on a national level (e.g. DVG guidelines in Germany); however, internationally accepted certification procedures are currently lacking.

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Development and validation of a cell culture based assay for in vitro assessment of anticryptosporidial compounds

Cited by 24 publications

References 23 publications

Effects of curcumin on Cryptosporidium parvum in vitro

Effects of curcumin on Cryptosporidium parvum in vitro

In vitro determination of anticryptosporidial activity of phytogenic extracts and compounds

Inactivation of exogenous endoparasite stages by chemical disinfectants: current state and perspectives

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