Effects of dietary water-soluble extract of rosemary supplementation on growth performance and intestinal health of broilers infected with Eimeria tenella.

This study was conducted to explore the effect of dietary supplementation of water-soluble extract of rosemary (WER) on growth performance and intestinal health of broilers infected with Eimeria tenella (E. tenella), and evaluate the anticoccidial activity of WER. 360 1-d-old Chinese indigenous male yellow-feathered broiler chickens were randomly allocated to six groups: blank control (BC) group and infected control (IC) group received a basal diet; positive control (PC) group, received a basal diet supplemented with 200 mg/kg diclazuril; WER100, WER200, and WER300 groups received a basal diet containing 100, 200, and 300 mg/kg WER, respectively. On day 21, all birds in the infected groups (IC, PC, WER100, WER200, and WER300) were orally gavaged with 1 mL phosphate-buffered saline (PBS) of 8 × 104 sporulated oocysts of E. tenella, and birds in the BC group were administrated an aliquot of PBS dilution. The results showed that dietary supplementation of 200 mg/kg WER increased the average daily gain of broilers compared to the IC group from days 22 to 29 (P < 0.001). The anticoccidial index values of 100, 200, and 300 mg/kg WER were 137.49, 157.41, and 144.22, respectively, which indicated that WER exhibited moderate anticoccidial activity. Compared to the IC group, the groups supplemented with WER (100, 200, and 300 mg/kg) significantly lowered fecal oocyst output (P < 0.001) and cecal coccidia oocysts, alleviated intestinal damage and maintained the integrity of intestinal epithelium. Dietary supplementation with WER significantly improved antioxidant capacity, elevated the levels of secretory immunoglobulin A, and diminished inflammation within the cecum, particularly at a dosage of 200 mg/kg. The results of this study indicated that dietary supplementation with 200 mg/kg WER could improve broiler growth performance and alleviate intestinal damage caused by coccidiosis.

Avian coccidiosis, a prevalent parasitic disease caused by Eimeria protozoa, leads to significant economic losses in the global poultry industry. Currently, the control of coccidiosis in chickens primarily relies on chemical and ionophore anticoccidials. However, the long-term use of these compounds has resulted in the development of drug-resistant strains, presenting a critical challenge. Additionally, the toxic and side effects of ionophore anticoccidials have become increasingly apparent. Thus, there is an urgent need to find economical and environmentally friendly measures to control coccidiosis in chickens. In this study, we established a model of Eimeria tenella infection in broilers to explore whether the water-soluble extract of rosemary (WER) could serve as an alternative method for controlling avian coccidiosis. Our results showed that dietary supplementation with WER (100, 200, and 300 mg/kg) had a beneficial anticoccidial effect, alleviating intestinal damage caused by coccidiosis by enhancing the intestinal antioxidant defense and activating the immune function of the infected broilers. Specifically, dietary supplementation with 200 mg/kg WER emerged as a promising strategy for controlling avian coccidiosis in the poultry industry.

The in vitro and in vivo anti-virulent effect of organic acid mixtures against Eimeria tenella and Eimeria bovis.

Eimeria tenella and Eimeria bovis are complex parasites responsible for the condition of coccidiosis, that invade the animal gastrointestinal intestinal mucosa causing severe diarrhoea, loss of appetite or abortions, with devastating impacts on the farming industry. The negative impacts of these parasitic infections are enhanced by their role in promoting the colonisation of the gut by common foodborne pathogens. The aim of this study was to test the anti-Eimeria efficacy of maltodextrin, sodium chloride, citric acid, sodium citrate, silica, malic acid, citrus extract, and olive extract individually, in vitro and in combination, in vivo. Firstly, in vitro infection models demonstrated that antimicrobials reduced (p < 0.05), both singly and in combination (AG), the ability of E. tenella and E. bovis to infect MDBK and CLEC-213 epithelial cells, and the virulence reduction was similar to that of the anti-coccidial drug Robenidine. Secondly, using an in vivo broiler infection model, we demonstrated that AG reduced (p = 0.001) E. tenella levels in the caeca and excreted faeces, reduced inflammatory oxidative stress, improved the immune response through reduced ROS, increased Mn-SOD and SCFA levels. Levels of IgA and IgM were significantly increased in caecal tissues of broilers that received 0.5% AG and were associated with improved (p < 0.0001) tissue lesion scores. A prophylactic approach increased the anti-parasitic effect in vivo, and results indicated that administration from day 0, 5 and 10 post-hatch reduced tissue lesion scores (p < 0.0001) and parasite excretion levels (p = 0.002). Conclusively, our in vitro and in vivo results demonstrate that the natural antimicrobial mixture (AG) reduced parasitic infections through mechanisms that reduced pathogen virulence and attenuated host inflammatory events.

Eukaryotic elongation factor 2 is involved in the anticoccidial action of diclazuril in the second-generation merozoites of Eimeria tenella.

Eimeria tenella, an obligate intracellular parasite, can actively invade the cecal epithelial cells of chickens and cause severe enteric disease. Eukaryotic elongation factor 2 (eEF2) plays a major role in protein synthesis and cell survival. This study aims to explore the exact mechanisms underlying diclazuril inhibition in second-generation merozoites of E. tenella. The eEF2 cDNA of the second-generation merozoites of E. tenella (EtEF2) was cloned by reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Diclazuril-induced expression profiles of EtEF2 were also analyzed. The cloned full-length cDNA (2893 bp) of the EtEF2 nucleotide sequence encompassed a 2499 bp open reading frame (ORF) that encoded a polypeptide of 832 residues with an estimated molecular mass of 93.12 kDa and a theoretical isoelectric point of 5.99. The EtEF2 nucleotide sequence was submitted to the GenBank database with the accession number KF188423. The EtEF2 protein sequence shared 99 % homology with the eEF2 sequence of Toxoplasma gondii (GenBank XP_002367778.1). The GTPase activity domain and ADP-ribosylation domain were conserved signature sequences of the eEF2 gene family. The changes in the transcriptional and translational levels of EtEF2 were detected through quantitative real-time PCR and Western blot analyses. The mRNA expression level of EtEF2 was 2.706 fold increases and the protein level of EtEF2 was increased 67.31 % under diclazuril treatment. In addition, the localization of EtEF2 was investigated through immunofluorescence assay. Experimental results demonstrated that EtEF2 was distributed primarily in the cytoplasm of second-generation merozoites, and its fluorescence intensity was enhanced after diclazuril treatment. These findings indicated that EtEF2 may have an important role in understanding the signaling mechanism underlying the anticoccidial action of diclazuril and could be a promising target for novel drug exploration.

The effect of autophagy on the survival and invasive activity of Eimeria tenella sporozoites.

Autophagy is a cellular process that is vital for the maintenance of homeostasis in eukaryotic cells. Currently, autophagy-related genes (atgs) in the Eimeria tenella genome database have been reported, but very little is known about the effects of autophagy on the survival and invasive activity of this protozoan. In this study, we investigated the autophagy in E. tenella sporozoites under starvation and autophagy-modulators treatments and evaluated the autophagy influence on cellular adenosine triphosphate (ATP) levels, the survival rate and the invasive activity of the sporozoites. The results showed that the autophagy could be induced in the sporozoites by starvation or inducer rapamycin (RP), but it could be inhibited by 3-methyladenine (3-MA) treatment. The sporozoites after starvation and RP-treatment displayed punctate signals of EtATG8 and formed autophagosomes. The survival rate of the sporozoites under starvation was significantly lower than that in the control group, whereas the ATP levels in sporozoite were far greater than those in the control. The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) showed that the invasive activity of the sporozoites was up- and down-regulated by RP and 3-MA induction, respectively. Our results indicate that autophagy has effects on the survival and invasive activity of E. tenella sporozoites, which may provide new insights into anti-coccidial drugs.

Anticoccidial effects of areca nut (Areca catechu L.) extract on broiler chicks experimentally infected with Eimeria tenella.

To study the anticoccidial effects of areca nut extract (ANE) on coccidiosis, 270 one-day old Wenchang broiler chicks were divided into six equal groups, each with three replicate cages (n = 15 per cage). The six groups were the blank control group (BC), negative control group (NC), positive control group (PC), and three ANE-treated groups. The birds in the three control groups (BC, NC and PC) were fed a basal diet without ANE supplementation. The birds in the three ANE-treated groups were fed a basal diet supplied with ANE at 100 (T1), 200 (T2), or 300 (T3) mg/kg feed. At 15 days of age, the birds in the NC, PC and the three ANE groups were challenged orally with 1 × 105Eimeria tenella oocysts per chick. At 48 h after oocysts inoculation, the birds in group PC were supplied diclazuril with drinking water for 5 days. The results showed that ANE and diclazuril significantly improved feed intake and body weight gain (P < 0.05) relative to the NC group. Both ANE and diclazuril significantly (P < 0.05) reduced OPG on day 4-9 post-inoculation (p.i.) relative to the NC group. Coccidial infection damaged the integrity of the cecal mucosa and thickened cecal tunica muscularis. ANE and diclazuril mitigated the mucosal damage caused by coccidial infection. Diet ANE supplementation reduced the cecal lesion scores compared to the NC group (P < 0.05). ANE and diclazuril increased nitric oxide (NO) levels at 3 days p.i., but reduced NO levels at 6 days p.i. (P < 0.05) compared to the NC group. Diet ANE supplementation increased the concentration of interleukin 2 (IL-2) in infected chicken relative to the NC group. The current results showed the anticoccidial properties, and beneficial effect on intestinal mucosa damage of ANE in broiler chicks challenged with coccidiosis.

Effect of ATP and Bax on the apoptosis of Eimeria tenella host cells.

BACKGROUND: Eimeria tenella (E. tenella) is a species of Eimeria that causes haemorrhagic caecal coccidiosis, resulting in major economic losses in the global poultry industry. After E. tenella infection, the amount of ATP and Bax in host cells showed highly significant changes. Therefore, it is necessary to investigate the effects of ATP and Bax on the apoptosis of E. tenella host cells. RESULTS: The ATP-treated group and the V5-treated group had higher E. tenella infection rates than the untreated group at 24, 48, 72, 96, and 120 h after infection with E. tenella. The results of flow cytometry showed that compared with the control group, the mitochondrial permeability transition pore (MPTP) opening in the untreated group was highly significantly increased (P < 0.01) at 4, 24, 48, 72, 96, and 120 h. Moreover, results from Hoechst-Annexin V-PI staining and flow cytometry showed that the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were significantly lower (P < 0.05) or highly significantly lower (P < 0.01) than those of the control group at 4 h, while the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were higher at varying degrees than those in the control group at 24-120 h (P < 0.05 or P < 0.01). After treatment with ATP and Bax inhibitors, the rates of early apoptosis, late apoptosis, and necrosis, in addition to the MPTP opening in both the ATP-treated and V5-treated groups, were significantly lower (P < 0.05) or highly significantly lower (P < 0.01) than those in the untreated group. CONCLUSIONS: ATP and Bax play important roles in regulating the apoptosis of E. tenella host cells.

Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.

The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.

Combination of cell culture and qPCR to assess the efficacy of different anticoccidials on Eimeria tenella sporozoites.

Three in vitro studies were designed to develop an assay for anticoccidial efficacy by use of laboratory (Houghton) and field (T-376) Eimeria tenella strains. In study (1), minimum inhibitory concentrations (MICs) of monensin (Mon), maduramicin (Mad), salinomycin (Sal), and lasalocid (Las) were determined that are able to inhibit more than 50% of sporozoites in host cell (Madin-Darby bovine kidney (MDBK)) penetration and more than 95% of Houghton sporozoites development to mature merozoites (treatment time 24 h) using quantitative real-time PCR (qPCR). MICs were 0.5, 2.5, 1, and 0.5 µg/ml for Mon, Mad, Sal, and Las, respectively. Applying the previous MIC on T-376 strain revealed a different sensitivity profile. Mad reduced T-376 gene copies by only 89.3% after 96 h of infection. In study (2), Houghton strain sporozoites were incubated with MIC of the different tested ionophores for 2 and 4 h, respectively; afterwards, their ability to invade MDBK cells was determined using phase-contrast microscopy and qPCR. Treatment of sporozoites with ionophores for 4 h resulted in significant inhibition of invasion compared with non-treated parasites as assessed both by microscopy as well as qPCR. Inhibition rates for Mon, Mad, Sal, and Las were 90.2, 75.0, 88.3, and 82.6% using phase-contrast microscopy and 83.9, 81.4, 85.8, and 75.4% using qPCR, respectively. T-376 sporozoite invasion into MDBK cells was reduced to 48.9% by Mad. Study (3) was conducted to determine inhibition exerted by toltrazuril (Tol). Tol at 5 µg/ml reduced reproduction of Houghton strain by 95%, whereas T-376 was only reduced by 86.5%. The presented experiments indicate that infectivity inhibition of sporozoites incubated for 4 h with anticoccidials and development inhibition after 96 h of infection by qPCR are suitable means to assess sensitivity of E. tenella strains to anticoccidials.

Reduced parasite motility and micronemal protein secretion by a p38 MAPK inhibitor leads to a severe impairment of cell invasion by the apicomplexan parasite Eimeria tenella.

E. tenella infection is associated with a severe intestinal disease leading to high economic losses in poultry industry. Mitogen activated protein kinases (MAPKs) are implicated in early response to infection and are divided in three pathways: p38, extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). Our objective was to determine the importance of these kinases on cell invasion by E. tenella. We evaluated the effect of specific inhibitors (ERK: PD98059, JNKII: SP600125, p38 MAPK: SB203580) on the invasion of epithelial cells. Incubation of SP600125 and SB203580 with epithelial cells and parasites significantly inhibited cell invasion with the highest degree of inhibition (90%) for SB203580. Silencing of the host p38α MAPK expression by siRNA led to only 20% decrease in cell invasion. In addition, when mammalian epithelial cells were pre-treated with SB203580, and washed prior infection, a 30% decrease in cell invasion was observed. This decrease was overcome when a p38 MAPK activator, anisomycin was added during infection. This suggests an active but limited role of the host p38 MAPK in this process. We next determined whether SB203580 has a direct effect on the parasite. Indeed, parasite motility and secretion of micronemal proteins (EtMIC1, 2, 3 and 5) that are involved in cell invasion were both decreased in the presence of the inhibitor. After chasing the inhibitor, parasite motility and secretion of micronemal proteins were restored and subsequently cell invasion. SB203580 inhibits cell invasion by acting partly on the host cell and mainly on the parasite.

Ethanol and isopropanol trigger rapid egress of intracellular Eimeria tenella sporozoites.

Egress from host cells is a vital step of the intracellular life cycle of apicomplexan parasites such as Toxoplasma gondii. This phenomenon has attracted attentions from many research groups. Previous studies have shown that ethanol could stimulate the release of microneme proteins by elevating intracellular Ca(2+) concentration of T. gondii, resulting in the parasite egress from host cells. However, little information about egress is known on Eimeria species, the causative agent of coccidiosis in poultry and livestock. In this report, we studied the effect of ethanol and isopropanol on the egress of eimerian parasites. Eimeria tenella sporozoites cultured in primary chicken kidney cells were treated with ethanol and isopropanol, then the egressed parasites were analyzed. Ethanol and isopropanol could induce the rapid egress of E. tenella sporozoites from host cells. No substantial damage was found in parasite-egressed host cells. Compared to the freshly isolated sporozoites, the re-invading ability and reproductivity of the egressed parasites significantly decreased by 43.4 and 44.1 % individually. We also found that fewer sporozoites egressed from host cells when the parasites developed for a longer time before the alcohol treatment. These results demonstrate an in vitro egress mode different from that of T. gondii, facilitating the deciphering of the mechanisms of egress of eimerian parasites.

Aminopeptidase N1 (EtAPN1), an M1 metalloprotease of the apicomplexan parasite Eimeria tenella, participates in parasite development.

Aminopeptidases N are metalloproteases of the M1 family that have been reported in numerous apicomplexan parasites, including Plasmodium, Toxoplasma, Cryptosporidium, and Eimeria. While investigating the potency of aminopeptidases as therapeutic targets against coccidiosis, one of the most important avian diseases caused by the genus Eimeria, we identified and characterized Eimeria tenella aminopeptidase N1 (EtAPN1). Its inhibition by bestatin and amastatin, as well as its reactivation by divalent ions, is typical of zinc-dependent metalloproteases. EtAPN1 shared a similar sequence, three-dimensional structure, and substrate specificity and similar kinetic parameters with A-M1 from Plasmodium falciparum (PfA-M1), a validated target in the treatment of malaria. EtAPN1 is synthesized as a 120-kDa precursor and cleaved into 96-, 68-, and 38-kDa forms during sporulation. Further, immunolocalization assays revealed that, similar to PfA-M1, EtAPN1 is present during the intracellular life cycle stages in both the parasite cytoplasm and the parasite nucleus. The present results support the hypothesis of a conserved role between the two aminopeptidases, and we suggest that EtAPN1 might be a valuable target for anticoccidiosis drugs.

Pharmacodynamic evaluation of a reference and a generic toltrazuril preparation in broilers experimentally infected with Eimeria tenella or E. acervulina.

Abstract 1. The aim of this study was to investigate the effects on pigmentation, faecal oocyst output, immune responsiveness and reactive oxygen species (ROS) generation following treatment with either the reference toltrazuril (Baycox) or a generic preparation (gen-TTZ), during an experimental Eimeria tenella (Et) or E. acervulina (Ea) infection of 210 Ross broiler chickens. 2. Results showed a significant difference on the anticoccidial efficacy 6 d after treating infected animals with Baycox (Et: 99.69% and Ea: 99.52%) or gen-TTZ (Et:85.71% and Ea 81.81%). 3. Gen-TTZ-treated animals were less strongly carotenoid-pigmented than Baycox-treated broilers. Mean plasma carotenoid concentrations were significantly higher in groups treated with Baycox than in broilers given gen-TTZ. 4. Treatment of animals with Baycox led to a significant decrease in ability of the peripheral blood mononuclear cells to produce ROS in contrast to gen-TTZ-treated groups. Baycox, but not generic toltrazuril, increased IL-10 and decreased tumour necrosis factor alpha (TNF-α) concentrations in chickens infected with E. tenella and E. acervulina. 5. It is suggested that differences in anticoccidial efficacy may be observed when using a generic toltrazuril product. Hence, in addition to plasma profiles of drugs, standardised clinical control tests may be necessary for generic formulations, particularly if other parameters are important to achieve a better control of coccidiosis.

Parasiticidal activity of a novel synthetic peptide from the core α-helical region of NK-lysin.

NK-lysin is an anti-microbial peptide that plays a critical role in innate immunity against infectious pathogens through its selective membrane disruptive property. We previously expressed and purified a full-length chicken NK-lysin (cNKL) recombinant protein, and demonstrated its in vitro anti-parasitic activity against the apicomplexan protozoan, Eimeria, the etiologic agent of avian coccidiosis. This study evaluated the in vitro and in vivo anti-parasitic properties of a synthetic peptide (cNK-2) incorporating a predicted membrane-permeating, amphipathic α-helix of the full-length cNKL protein. The cNK-2 peptide exhibited dose- and time-dependent in vitro cytotoxic activity against E. acervulina and E. tenella sporozoites. The cytotoxic activity of 1.5 µM of cNK-2 peptide against E. acervulina following 6h incubation was equal to that of 2.5 µM of melittin, the principal active component of apitoxin (bee venom) that also exhibits anti-microbial activity. Even greater activity was detected against E. tenella, where 0.3 µM of cNK-2 peptide was equivalent to 2.5 µM of melittin. Against Neospora caninum tacyzoites, however, the cytotoxic activity of cNK-2 peptide was inferior to that of melittin. Transmission electron microscopy of peptide-treated E. tenella sporozoites revealed disruption of the outer plasma membrane and loss of intracellular contents. In vivo administration of 1.5 µM of cNK-2 peptide increased protection against experimental E. acervulina infection, as measured by greater body weight gain and reduced fecal oocyst shedding, compared with saline controls. These results suggest that the cNK-2 synthetic peptide is a novel anti-infective peptide that can be used for protection against avian coccidiosis during commercial poultry production.

A novel serine/threonine protein phosphatase type 5 from second-generation merozoite of Eimeria tenella is associated with diclazuril-induced apoptosis.

Screening the anticoccidial drug targets is very important for developing novel drugs and revealing the molecular basis of drug resistance in coccidia. Due to high effectivity and safety, diclazuril was used widely in the poultry industry. To assess the roles of the serine/threonine protein phosphatase type 5 of second-generation merozoites in Eimeria tenella (EtPP5) in the anticoccidial activity of diclazuril against chicken coccidiosis, EtPP5 was cloned using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Ultrastructural changes in second-generation merozoites and mRNA expression level of EtPP5 were monitored by transmission electron microscopy (TEM) and quantitative real-time PCR, respectively. The results showed that the full length of the cloned EtPP5 cDNA (2,495 bp) encompassed a 1,647-bp open reading frame encoding a polypeptide of 548 residues with an estimated molecular mass of 60.82 kDa and a theoretical isoelectric point of 5.89. Molecular analysis of EtPP5 reveals the presence of a C-terminal phosphatase domain and an extended N-terminal tetratricopeptide repeat motif, a typical feature of protein phosphatases. The cDNA sequence has been submitted to the GenBank database with accession number JX987508. EtPP5 shared 89% homology with the published sequence of a PP5 ortholog of Toxoplasma gondii at the amino acid level (GenBank XP_002364442.1). TEM observed that diclazuril induced ultrastructural changes in second-generation merozoites. Quantitative real-time PCR analysis showed that compared with the control group, the level of EtPP5 mRNA expression was significantly downregulated by 51.4% by diclazuril treatment. The high similarity of EtPP5 to previously described PP5 of other organisms, as well as its downregulated expression and connection with apoptosis in the second-generation merozoites induced by diclazuril, suggests that it could act an important role in understanding the signaling mechanism underlining the diclazuril-induced merozoites apoptosis.

In vitro inhibition of Eimeria tenella invasion of epithelial cells by phytochemicals.

Resistance to coccidiostats and possible future restrictions on their use raise the need for alternative methods of reducing coccidiosis in poultry. The aim of this study was to evaluate the effect of selected phytochemicals on Eimeria tenella sporozoite invasion in vitro. Four phytochemicals were selected on the basis that they reduce the virulence of Eimeria spp. and/or provide immune modulatory benefits to host cells: betaine, carvacrol, curcumin and Echinacea purpurea extract (EP). Madin-Darby bovine kidney (MDBK) cells were covered by medium containing phytochemicals at the highest concentration which was non-toxic to the cells. Salinomycin 50 µg/ml was positive control; negative control was medium only. E. tenella (Houghton strain) sporozoites were added to wells and after incubation for 2, 4 or 20 h at 37°C, cells were fixed and stained with hematoxylin-eosin. Ten evenly spaced fields per well were photographed and the percentage of cells invaded by sporozoites was calculated and normalized to the control. At 2h, carvacrol, curcumin and EP showed a significantly lower percentage of sporozoite invasion than the untreated control; in contrast, betaine treatment represented a significantly higher invasion percentage. Combining carvacrol with EP inhibited E. tenella invasion more effectively than applying the compounds individually, but the further addition of curcumin did not reduce invasion further. In conclusion, this study shows that invasion of MDBK epithelial cells by E. tenella sporozoites is inhibited in the presence of carvacrol, curcumin, or EP and enhanced by betaine. There may be potential for developing these phytochemicals as anti-coccidial feed or water additives for poultry.

Identification of lead compounds targeting the cathepsin B-like enzyme of Eimeria tenella.

Cysteine peptidases have been implicated in the development and pathogenesis of Eimeria. We have identified a single-copy cathepsin B-like cysteine peptidase gene in the genome database of Eimeria tenella (EtCatB). Molecular modeling of the predicted protein suggested that it differs significantly from host enzymes and could be a good drug target. EtCatB was expressed and secreted as a soluble, active, glycosylated mature enzyme from Pichia pastoris. Biochemical characterization of the recombinant enzyme confirmed that it is cathepsin B-like. Screening of a focused library against the enzyme identified three inhibitors (a nitrile, a thiosemicarbazone, and an oxazolone) that can be used as leads for novel drug discovery against Eimeria. The oxazolone scaffold is a novel cysteine peptidase inhibitor; it may thus find widespread use.

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.

Eimeria tenella glucose-6-phosphate isomerase: molecular characterization and assessment as a target for anti-coccidial control.

Limitations with current chemotherapeutic and vaccinal control of coccidiosis caused by Eimeria species continue to prompt development of novel controls, including the identification of new drug targets. Glucose-6-phosphate isomerase (G6-PI) has been proposed as a valid drug target for many protozoa, although polymorphism revealed by electrophoretic enzyme mobility has raised doubts for Eimeria. In this study we identified and sequenced the Eimeria tenella G6-PI orthologue (EtG6-PI) from the reference Houghton strain and confirmed its position within the prevailing taxonomic hierarchy, branching with the Apicomplexa and Plantae, distinct from the Animalia including the host, Gallus gallus. Comparison of the deduced 1647 bp EtG6-PI coding sequence with the 9016 bp genomic locus revealed 15 exons, all of which obey the intron-AG-/exon/-GT-intron splicing rule. Comparison with the Weybridge and Wisconsin strains revealed the presence of 33 single nucleotide polymorphisms (SNPs) and 14 insertion/deletion sites. Three SNPs were exonic and all yielded non-synonymous substitutions. Preliminary structural predictions suggest little association between the coding SNPs and key G6-PI catalytic residues or residues thought to be involved in the coordination of the G6-PI's substrate phosphate group. Thus, the significant polymorphism from its host orthologue and minimal intra-specific polymorphism suggest G6-PI remains a valid anti-coccidial drug target.

Influence of monensin on cation influx and Na+ -K+ -ATPase activity of Eimeria tenella sporozoites in vitro.

The experiments were conducted to determine intrasporozoite Na+/K+ concentrations (by AAS) and membrane-bound Na+ -K+ -ATPase activity (measured by UV-VIS with a Na+ -K+ -ATPase Detection Kit) of Eimeria tenella sporozoites of the sensitive line (i.e., the parent line, coded as OS) and 2 resistant lines, derived from the parent line (coded as OR125 and OR200), with and without in vitro exposure to monensin. These parameters for OR125 and OR200 were significantly lower than those for OS. In vitro exposure to monensin increased intrasporozoite Na+/K+ concentrations and Na+ -K+ -ATPase activity, but the stimulation on OS was significantly higher than those on OR125 and OR200, indicating that monensin had less effect on resistant parasites. The results of this study suggest that altered biochemical or physiological properties, or both, in the membranes of E. tenella might be related to a reduced sensitivity to monensin.

Synthesis and SAR studies of very potent imidazopyridine antiprotozoal agents.

Compounds 10a (IC50 110 pM) and 21 (IC50 40 pM) are the most potent inhibitors of Eimeria tenella cGMP-dependent protein kinase activity reported to date and are efficacious in the in vivo antiparasitic assay when administered to chickens at 12.5 and 6.25 ppm levels in the feed. However, both compounds are positive in the Ames microbial mutagenesis assay which precludes them from further development as antiprotozoal agents in the absence of negative lifetime rodent carcinogenicity studies.