In vitro determination of anticryptosporidial activity of phytogenic extracts and compounds

Abstract

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.

In vitro anthelmintic assessment of selected phytochemicals against Hymenolepis diminuta, a zoonotic tapeworm

“…Phytochemicals possess a wide range of chemical structures, and in experimental studies, different phytocompounds have also shown an array of biological activities, which have often been found to vary from compound to compound (Newman and Cragg 2007;Teichmann et al 2012;Wink 2012). This may explain, in part, as to why, in the present study, different tested phytochemicals revealed different levels of anthelmintic activity.…”

“…Although, there are many evidences of the antiparasitic effects of these phytoconstituents, however, their purported antiparasitic effects have also been the cause of controversy amongst the scientific community (Athanasiadou and Kyriazakis 2004). A perusal of literature reveals that majority of scientific evidences supporting the antiparasitic properties of phytoconstituents have come from in vitro studies (Athanasiadou and Kyriazakis 2004;Nagajyothi et al 2012;Teichmann et al 2012;Zahir et al 2012), because employing this method the phytoconstituents can be reliably quantified without the interference of other plant components or nutrients. Therefore, in this study we employed a suitable in vitro assay to test the anthelmintic efficacy of four selected phytochemicals on H. diminuta.…”

“…Plants produce a high diversity of low molecular weight compounds and in recent years some of these phytocompounds have also shown good antiparasitic effects in laboratory trials (Anthony et al 2005;Kumar et al 2012;Nagajyothi et al 2012;Teichmann et al 2012;Zahir et al 2012;González-Coloma et al 2012;Koné et al 2012). The Northeast region of India is blessed with a rich wealth of medicinal plants, whose decoctions, infusions, syrups, etc.…”

Anti-parasitic activity of polyether ionophores

“…parvum both in vitro and in vivo [177][178][179][180][181][182][183][184][185][186], among which maduramicin and monensin (Figure 1) were identified as the most active ones [177][178][179][180][183][184][185][186]. µM without any adverse effects to the host cells [178].…”

In vitroinhibitory effects of plant-derived by-products againstCryptosporidium parvum

“…Plants present a rich source of bioactive compounds and have a long history of use for prevention and treatment of various human and animal illnesses, including parasitic diseases [ 1 , 20 , 29 , 31 ]. For example, the isoflavone genistein from soybean, the flavonolignan mix silymarin (with silibinin as a main compound) from the milk thistle [ 22 , 39 , 43 ] or the ferulic acid-derived curcuminoid curcumin from turmeric [ 34 ] have been found to possess anti-cryptosporidial activity in vitro . The xanthone mangiferin (known from mango) has demonstrated some efficacy in vivo [ 30 ].…”