Effects of curcumin (diferuloylmethane) on Eimeria tenella sporozoites in vitro.

The negative effects of coccidiosis on poultry health and productivity and increasing problems related to drug resistance have stimulated the search for novel and alternative methods of control. The present study evaluates the anticoccidial activity of curcumin (diferuloylmethane), a natural polyphenolic compound abundant in the rhizome of the perennial herb turmeric (Curcuma longa) which is a spice and food colorant commonly used in curries and also used as medicinal herb. Its effects were evaluated on Eimeria tenella sporozoites, including morphological alterations, sporozoite viability and infectivity to Madin-Darby bovine kidney (MDBK) cells. Morphological alterations of the sporozoites were recorded as deformation due to swelling and cell membrane corrugations. Curcumin at concentrations of 25, 50, 100, 200 and 400 µM showed considerable effects on sporozoite morphology and viability in a dose-dependent manner after incubation over 3, 6, 18 and 24 h while lower curcumin concentrations (6.25 and 12.5 µM) were not effective. In comparison to the untreated control, sporozoite infectivity was reduced at curcumin concentrations of 100 and 200 µM by 41.6% and 72.8%, respectively. Negative effects of curcumin on MDBK cells were not seen at these concentrations; however, curcumin at concentrations of 1,800, 600 and 400 µM was toxic to MDBK cells and affected cell proliferation. In conclusion, curcumin exhibited a marked inhibitory effect in vitro on E. tenella sporozoites inducing morphological changes and reducing sporozoite viability and infectivity.

In vitro study of anti-coccidial activity of essential oils from indigenous plants against Eimeria tenella.

This study was designed to evaluate the in vitro anticoccidial properties against Eimeria tenella of different essential oils and their major active components. Efficacy of ten essential oils from different Thai indigenous plants were preliminarily screened and only those with potential were further tested for effective concentrations and identifying their active compounds. Oocysticidal property was evaluated in term of sporulation inhibition of oocysts and the percentage of unsporulated, sporulated and degenerated oocysts, after treatment with 125µg/ml of the selected essential oil, the sample was enumerated by haemocytometer, while coccidiocidal activity was assessed by the inhibition of sporozoite invasion in MDBK cell lines. Results showed that only Boesenbergia pandurata and Ocimum basilicum essential oils had strong sporulation inhibition activity by providing a higher ratio of degenerated oocysts and their IC50 were 0.134 and 0.101mg/ml, respectively. GC-MS analysis of B. pandurata essential oil found trans-b-ocimene, camphor, 1,8-cineole, geraniol, camphene, methyl cinnamate, l-limonene and linalool as the major components, while methyl chavicol, α-bergamotene, 1,8-cineole and trans-ß-ocimene were the main compounds of O. basilicum essential oil. Methyl cinnamate and camphor were the active components of B. pandurata oil, whereas methyl chavicol was the active component of O. basilicum oil by exhibiting the oocysticidal effect against E. tenella with IC50 values of 0.008, 0.023 and 0.054mg/ml, respectively. Furthermore, B. pandurata and O. basilicum oils also showed a strong cytotoxic property against coccidia with more than 70% inhibition of sporozoite invasion in MDBK cell lines, and their IC50 were 0.004 and 0.004mg/ml, respectively. Methyl cinnamate as well as camphor from B. pandurata and methyl chavicol from O. basilicum were also effective with IC50 values of 0.029, 0.023, and 0.022mg/ml, respectively.

Synthesis of apicidin-derived quinolone derivatives: parasite-selective histone deacetylase inhibitors and antiproliferative agents.

Apicidin's indole was efficiently converted into a series of N-substituted quinolone derivatives by indole N-alkylation followed by a two-step, one-pot, ozonolysis/aldol condensation protocol. The new quinolones exhibited good parasite selectivity and potency both at the level of their molecular target, histone deacetylase, and in their whole cell antiproliferative activity in vitro.

A novel method for counting eimerian oocysts at very low concentrations in aqueous suspensions.

A novel method for counting eimerian oocysts in samples of drinking water has been developed to fulfil the need for monitoring the delivery of very low concentrations of live anticoccidial vaccines to poultry via pipeline nipple-drinker systems. Advantages of the method are the ease of sample collection and processing, high degrees of accuracy and precision, and a sensitivity of one oocyst ml(-1). Results of a validation test are presented, with a protocol for the method and notes on its use. The coefficient of variation (CoV) of 10 sets of oocyst counts with nominal means of 10 to 160 oocysts ml(-1)ranged from about 16 per cent down to 6 per cent. The recovery efficiency for all 100 validation counts averaged 100.2 per cent with a range of 70-130 per cent. A practical example of field use of the method is given, including a modification to decrease the time taken for counting. In this case, when oocysts were pumped around a pipeline circuit of 129 m for 2.5 hours, the CoV of a mean of 112 oocysts ml(-1)(n = 10) was 12.4 per cent.

High-throughput RNA sequencing profiles and transcriptional evidence of aerobic respiratory enzymes in sporulating oocysts and sporozoites of Eimeria tenella.

Seven species of Eimeria are responsible for coccidiosis in chickens. Eimeria tenella is one of the most pathogenic parasites since it is associated with high mortality and great economic impact. The life cycle of the parasite includes development in the environment and in the intestinal tract. We conducted RNA sequencing using a next generation sequencer to obtain transcriptome information from the sporulating oocysts, and sporozoites. We collected 2.8 million 75 bp reads of a short-tag sequence, and 25,880 contigs were generated by the Oases assembler. A Blastx search of GenBank databases revealed that 7780 contigs (30.1%) had significant homology with deposited sequence data (E-value <1e-6); among these contigs, 6051 contigs were similar to those of Toxoplasma gondii while only 513 contigs (6.6%) were similar to those of E. tenella. After an orthological analysis conducted with the UniProt database of T. gondii, 6661 contigs were distributed within the categories of cellular components (1528 gene categories), biological processes (861 gene categories), and molecular functions (241 gene categories). The significantly matched contigs contained high numbers of enzymes associated with glycolysis, TCA, and the pentose-phosphate pathway. Most of the enzymes, measured by quantitative reverse transcription-PCR, were up-regulated in sporulating stage. These results suggest that the intracellular carbohydrate amylopectin could be used as an energy source for ATP production including glycolysis and the pentose-phosphate pathway, which generates NADPH and pentoses. Our data also suggest that Eimeria might possess a partial or similar pathway to the TCA cycle essential for aerobic respiration. Furthermore, the newly annotated and non-annotated contigs might contain E. tenella-specific or novel sequences.

piggyBac transposon-mediated transgenesis in the apicomplexan parasite Eimeria tenella.

piggyBac, a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella. Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5' and 3' ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac.

Location of RAD51-like protein during meiotic prophase in Eimeria tenella.

This study focuses on reporting events in Eimeria tenella oocysts from early to late prophase I in terms of RAD51 protein in association with the synaptonemal complex formed between homologous chromosomes. The aim of the study was the sequential localization of RAD51 protein, which is involved in the repair of double-strand breaks (DSBs) on the eimerian chromosomes as they synapse and desynapse. Structural Maintenance of Chromosome protein SMC3, which plays a role in synaptonemal complex formation, was labeled to identify initiation and progress of chromosome synapsis and desynapsis in parallel with the appearance and disappearance of RAD51 foci. Antibodies directed against RAD51 and cohesin subunit SMC3 proteins were labeled with either fluorescence or colloidal gold to visualize RAD51 protein foci and synaptonemal complexes. RAD51 protein localization during prophase I was studied on meiotic chromosomes spreads obtained from oocysts at different points in time after the start of sporulation. The present findings showed that foci detected with the antibody directed against RAD51 protein first appeared at the pre-leptotene stage before homologous chromosomes began pairing. Subsequently, the foci were detected in association with the lateral elements at the precise sites where synapsis were in progress. These findings lead us to suggest that in E. tenella, homologous chromosome pairing was a DSB-dependent mechanism and reinforced the participation of RAD51 protein in meiotic homology search, alignment and pairing of chromosomes.

Meiotic chromosome pairing and bouquet formation during Eimeria tenella sporulation.

In Eimeria tenella, meiotic division occurs exclusively in oocysts within the first 8h of sporulation. Difficulties with the wall-oocyst breakage in gaining access to chromosomes during meiosis have resulted in a scarcity of morphological data on Eimeria chromosomes. This study tracks the general behaviour of telomeres, attachment plaques and synaptonemal complexes in the nucleus of the meiotic oocyst of E. tenella. Fluorescence microscopy methods, in combination with immunoelectron microscopy techniques, were applied to obtain a series of time-lapse images during oocyst sporulation. Antibodies to Structural Maintenance of Chromosome proteins SMC1 and SMC3, and lamin were labelled with either fluorescence or colloidal gold to visualise the telomeres, central elements of the synaptonemal complex (SC) and nuclear periphery, respectively, at both the structural and ultrastructural levels. Using oocyst spreads and ultrathin sections of fixed oocysts it was possible to study telomere dynamics at stages during meiosis. The stages of the meiotic prophase I are delineated on the basis of the telomere position and the SC synapsis and desynapsis. During the leptotene stage, at 4h following the start of sporulation, meiotic chromosomes attached to the nuclear envelope. At that stage, chromosome synapsis was initiated in the telomeric regions but no interstitial synapsis pairing was observed. In the zygotene stage, telomere signals were clustered in a limited area of the nuclear envelope. Bouquet formation occurred at 5h after the start of sporulation, whereas chromosomes did not appear completely synapsed until the pachytene stage at 6h of sporulation. Desynapsis was observed at 8h of sporulation during the diplotene stage. This study provides the first morphological description of both the behaviour of the chromosomes and the timing of the prophase I stages in the meiotic nucleus of E. tenella.

A simple method for the purification of Eimeria tenella sporozoites.

A rapid, simple and cheap method for the purification of Eimeria tenella sporozoites has been developed using commercially available filter paper (595 filter paper circles, Schleicher & Schuell, 3354 Dassel, Germany, order 311610). Yield and purity are of the same value as by purification with a leukopac column (Bontemps & Yvore 1974). The described method can be used for the purification of Eimeria tenella sporozoites under sterile conditions for subsequent in vitro cultivation.