Anticoccidial activities of Piper betle L essential oil on Eimeria tenella oocysts.

Coccidiosis poses a significant threat to the poultry industry, with synthetic antibiotics and disinfectants being the primary tools for control. This study investigated the potential of Piper betle L essential oil (PBEO) as a natural alternative against Eimeria tenella, one of the most pathogenic Eimeria species affecting poultry. Our findings revealed that PBEO exhibits significant anticoccidial effects through two primary mechanisms: (i) oocysticidal activity by disintegrating oocyst walls and (ii) inhibition of the sporulation process. PBEO demonstrated oocysticidal activities ranging from 8.67 to 95.33% across concentrations from 0.04 to 40%. Notably, at 72 h post-incubation, a 0.04% PBEO concentration significantly reduced the number of sporulated oocysts (P ≤ 0.05) to 71.67%, showing effects comparable to those of formalin. PBEO reduced 50% of oocyst sporulation (IC50) in the concentration of 1.31% at 72 h. Gas chromatography-mass spectrometry (GC-MS) identified the primary constituents of PBEO, including eugenol, beta-caryophyllene, and other key compounds, collectively constituting 96% of the oil. This research underscores the potential of PBEO as a natural anticoccidial agent and lays the groundwork for further studies aimed at identifying, isolating, and developing active compounds that may specifically target the sporogony process in coccidian parasites.

Efficacy and safety of diclazuril nanoemulsion in control of Eimeria tenella in broilers.

BACKGROUND: Nanotechnology has the potential to reduce drug dosage while increasing efficacy; thus, the current work intends to synthesize diclazuril nanoemulsion and assess its performance against experimental coccidiosis in broilers. METHODS: Diclazuril nanoemulsion (DZN) was formulated and characterized by zeta seizer and zeta potential. The formulated DZN was evaluated in vivo against Eimeria tenella infected chicks. DZN and DZ were used in 2 programs; therapeutic and prophylactic. A total of 210 one-day-old broiler chicks were distributed equally into six groups. The controls were negative uninfected untreated and positive infected untreated (G1 & G2). Therapeutic groups (G3 & G4) treated by DZ and DZN after appearance of the clinical signs of coccidiosis and continued for 5 days. Prophylaxis groups (G5 & G6) received DZ and DZN at 3 days before challenge and continued for 5 days after infection. The treatments dosages were 10 mg/mL for DZ of commercial origin and 2.5 mg/mL for the prepared DZN. All groups (except negative control) orally infected then followed up for clinical signs of coccidiosis, mortality rate, oocysts count, performance, hematological and biochemical parameters in addition to histopathological lesions. RESULTS: The therapeutic groups showed that both treated groups (DZ and DZN) revealed similar results including good body weight gain, a low lesion caecal score, a low daily and total oocyst shedding count, and a low mortality rate. Regarding the biochemical parameters, all parameters were affected during infection then restored after the 12th day post infection. However, in the prophylactic groups, showed mild clinical signs and the blood pictures and biochemical parameters were nearly like the control negative without infection. CONCLUSION: DZN at a quarter dose of standard DZ produced the same outcomes as DZ at 10 mg/mL. Furthermore, DZN does not impair the typical safety of diclazuril in treated chicks.

Antioxidant properties of D-limonene and its nanoemulsion form enhance its anticoccidial efficiency in experimentally infected broilers with Eimeria tenella: an in vitro and in vivo study.

The excessive use of conventional medications to treat coccidiosis has led to concerns regarding drug residues in tissues and the emergence of multidrug resistance. Essential oils with anti-inflammatory and antioxidant activities may also have anticoccidial effects. The present study investigated the efficacy of D-limonene and its nanoemulsion form against Eimeria tenella in chickens. An in vitro study was conducted to evaluate the sporulation inhibitory effects of D-limonene on Eimeria tenella oocysts. Five D-limonene concentrations (0.625, 1.25, 2.5, 5, and 10% v/v) were tested alongside positive (10% formalin) and negative (2.5% potassium dichromate) controls. Each ELISA plate well was inoculated with 1200 unsporulated oocysts and incubated at 30 °C for 24, 48, and 72 h. Subsequently, samples were microscopically examined to assess sporulation inhibition and calculate the percentage of sporulated oocysts. For the in vivo study, 125 eight-day-old broiler chicks were divided into five groups of 25 birds each. The control negative group remained uninfected and untreated. The control positive group was challenged with 5 × 104 sporulated Eimeria tenella oocysts. The diclazuril group received 0.2 mg/kg diclazuril in their diet two days prior to, and until 10 days post infection. The D-limonene (DL) and D-limonene nanoemulsion (DLN) groups were challenged with 5 × 104 sporulated E. tenella oocysts at 18 days of age and administered 150 mg/L of their respective treatments in drinking water from day eight until the end of the experiment. Results from the in vitro study demonstrated that D-limonene suppressed oocyst sporulation by 50.83% at its highest concentration of 10%. In the in vivo study, both DL and DLN treated groups exhibited a significant reduction in oocyst output per gram of feces (OPG), along with increased body weight and decreased parasite stages in the cecal tissue. Furthermore, these treatments were associated with elevated levels of antioxidant enzymes such as glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD), accompanied by a decrease in malondialdehyde (MDA) and nitric oxide (NO) levels. Particularly, DLN treatment remarkably increased the number of goblet cells. In conclusion, D-limonene and its nanoemulsion represent promising alternatives for managing coccidiosis in poultry. They not only effectively control parasites but also promote intestinal health and boost antioxidant defenses.

Traditional Chinese medicine formulation ChangQing compound has significant therapeutic effects on chickens infected with Eimeria tenella.

Coccidiosis poses a significant challenge to the poultry industry. However, the excessive and improper use of anticoccidial drugs and vaccines has led to resistance and food safety concerns. Consequently, traditional Chinese herbs have garnered attention as a potentially safer and more effective alternative. ChangQing compound derived from various Chinese herbal medicines is a promising anticoccidiosis agent, but its therapeutic effects have not been comprehensively evaluated. This study aimed to assess the therapeutic efficacy of ChangQing Compound against Eimeria tenella-induced coccidiosis in chickens on the basis of physiological indicators, cecum lesions, and changes in microbial diversity. The comparison with the positive control group revealed the average weight gain (AWG) and anticoccidial index (ACI) of the chicks were significantly higher, in contrast, the feed conversion ratio (FCR), cecal lesion score (CLS), and oocyst count per gram of cecal content (OPG) were significantly lower (P<0.05). Notably, AWG (138.87 g), OPG (0.57 × 106), ACI (177.92), and FCR (2.51) reflected the significant therapeutic effect of the 2.5 g/L ChangQing compound treatment (CQM). Histological sections showed that the cecal villus damage and intestinal wall swelling were minimal in the CQM, consistent with the CLS (0.73). Additionally, the 2.5 g/L ChangQing compound treatment effectively prevented the decrease of red blood cells, platelets, and hemoglobin, while promoting the release of anti-inflammatory factors interleukin-10 and interleukin-4, and inhibiting the pro-inflammatory factors interferon-γ and interleukin-17. The microbial community structure in the CQM was most similar to that of the negative control group. In summary, ChangQing compound had multiple positive effects (e.g., promoting weight gain, alleviating anemia, suppressing coccidial proliferation, reducing intestinal damage, modulating immunity, and maintaining intestinal microbiota homeostasis). The study results may be relevant to developing a novel strategy for the clinical management of coccidiosis.

Anticoccidial activity of a botanical natural product based on eucalyptus, apigenin and eugenol against Eimeria tenella in broiler chickens.

BACKGROUND: Chicken coccidiosis is an intracellular parasitic disease that presents major challenges to the development of the commercial poultry industry. Perennial drug selective pressure has led to the multi-drug resistance of chicken coccidia, which makes the prevention and control of chicken coccidiosis extremely difficult. In recent years, natural plant products have attracted the attention of researchers due to their inherent advantages, such as the absence of veterinary drug residues. The development of these natural products provides a new direction for the prevention and treatment of chicken coccidiosis. METHODS: The anticoccidial effect of a natural plant product combination formulation (eucalyptus oil + apigenin + eugenol essential oil) was tested against Eimeria tenella in broilers. To search for the optimal concentration of the combination formulation, we screened 120 broilers in a chicken cage trial in which 100 broilers were infected with 5 × 104 sporulated Eimeria tenella oocysts; broilers receiving a decoquinate solution was set up as a chemical control. The optimal anticoccidial concentration was determined by calculating the anticoccidial index (ACI), and the suitable concentration was used as the recommended dose for a series of safety dose assessment tests, such as feed conversion ratio (FCR), hematological indices and serum biochemical indices, as well as liver and kidney sections, at onefold (low dose), threefold (medium dose) and sixfold (high dose) the recommended dose (RD). RESULTS: The results showed that this combination formulation of three plant natural products had a better anticoccidial effect than formulations containing two plant natural products or a single one, with an ACI of 169.3. The dose gradient anticoccidial test revealed that the high-dose formulation group had a better anticoccidial effect (ACI = 169.2) than the medium- and low-dose groups. The safety evaluation test showed that concentrations of the formulation at one-, three- and sixfold the RD were non-toxic to Arbor Acres broilers, indicating the high safety of the combination formulation. CONCLUSIONS: The combination formulation showed not only a moderate anticoccidial effect but also had a high safety profile for broilers. The results of this study indicate a new alternative for the prevention and control of coccidiosis in broilers.

Drug resistance and genetic characteristics of one Eimeria tenella isolate from Xiantao, Hubei Province, China.

Chicken coccidiosis causes retarded growth and low production performance in poultry, resulting in huge economic losses to the poultry industry. In order to prevent and control chicken coccidiosis, great efforts have been made to develop new drugs and vaccines, which require pure isolates of Eimeria spp. In this study, we obtained the Eimeira tenella Xiantao isolate by single oocyst isolation technology and compared its genome with the reference genome GCF_000499545.2_ETH001 of the Houghton strain. The results of the comparative genomic analysis indicated that the genome of this isolate contained 46,888 single nucleotide polymorphisms (SNPs). There were 15,107 small insertion and deletion variations (indels), 1693 structural variations (SV), and 3578 copy number variations (CNV). In addition, 64 broilers were used to determine the resistance profile of Xiantao strain. Drug susceptibility testing revealed that this isolate was completely resistant to monensin, diclazuril, halofuginone, sulfachlorpyrazine sodium, and toltrazuril, but sensitive to decoquinate. These data improve our understanding of drug resistance in avian coccidia.

Anticoccidial potentials of Azadirachta indica ethosomal nanovesicle in broiler chicks.

This study evaluated the efficacy of Azadirachta indica ethosomal nanovesicle against Eimeria tenella infection in broiler chicks. Azadirachta indica ethanolic extract was screened phtochemically and analyzed active components of the extracts using high­performance liquid chromatography (HPLC). Azadirachta indica ethosomal nanovesicle was synthesized and characterized by zeta potential and scanning electron microscope. Broiler chicks were allocated into seven groups. Control group. The second group administered nanosized ethosomal vesicles (1 mL/kg b.wt.). The third group administered Azadirachta indica nanovesicles (30 mg/kg b.wt.) from 10th day of age. Fourth group was infected with E. tenella at a dose of 1 mL containing 40000 oocyst/ chick at 14th day of age. The fifth group administered Azadirachta indica nanovesicle (30 mg/kg b.wt.) from 10th day of age and infected with E. tenella as fourth group. The sixth group infected with E. tenella as the fourth group and treated with Azadirachta indica nanovesicle (30 mg/kg b.wt. for 4 days after clinical signs appearance. The seventh group infected with E. tenella as the fourth group and treated with diclazuril group (1 mL/4 L of water) for 2 successive days. Coccidiosis significantly decreased body weight, feed intake, reduced glutathione (GSH) level while increased feed conversion ratio, oocyst count, malonaldehyde (MDA) and nitric oxide (NO) serum levels, protein expression of interleukin-1 beta (IL-1ß), interleukin 6 (IL-6), BAX and Caspase 3, in cecal tissue and induced cecal tissue injury. However, administration of coccidiosis chicks Azadirachta indica nanovesicle enhanced body weight, and serum GSH. While decreased feed intake, feed conversion ratio, oocyst count, MDA, and NO serum levels, and protein expression of IL-1ß, IL-6, BAX, and caspase 3 in cecal tissues and ameliorated cecal tissue damage. This study indicated that, A. indica ethosomal nanovesicle had potent anticoccidial properties.

Efficacy of liver free and Chitosan against Eimeria tenella in chickens.

Eimeria spp. are the pathogen that causes coccidiosis, a significant disease that affects intensively reared livestock, especially poultry. Anticoccidial feed additives, chemicals, and ionophores have routinely been employed to reduce Eimeria infections in broiler production. Therefore, the shift to antibiotic-free and organic farming necessitates novel coccidiosis preventive strategies. The present study evaluated the effects of potential feed additives, liver free and chitosan, against Eimeria tenella infection in White Leghorn broiler female chickens. One hundred sixty-five 1-day-old White Leghorn broiler female chicks were divided into 11 groups (15 female chicks per group), including the positive control group (G1), the negative control group (G2), a chitosan-treated group (G3), a chitosan-treated-infected group (G4), the liver free-treated group (G5), the liver free-treated-infected group (G6), the liver free-and-chitosan-treated group (G7), the liver free-and-chitosan-infected group (G8), the therapeutic liver free-and-chitosan-treated-infected group (G9), the sulfaquinoxaline-treated group (G10), and the sulfaquinoxaline-treated-infected group (G11). Chitosan was fed to the chicks in G3 and G4 as a preventative measure at a dose of 250 mg/kg. The G5 and G6 groups received 1.5 mg/kg of Liverfree. The G7 and G8 groups received chitosan and Liverfree. The G10 and G11 groups were administered 2 g/L of sulfaquinoxaline. From the moment the chicks arrived at Foshan University (one-day-old chicks) until the completion of the experiment, all medications were given to them as a preventative measure. G8 did; however, receive chitosan and liver free as therapeutic supplements at 7 dpi. The current study showed that the combination of liver free and chitosan can achieve better prophylactic and therapeutic effects than either alone. In E. tenella challenged chickens, G8 and G9 chickens showed reduced oocyst shedding and lesion score, improved growth performance (body weight, body weight gain, feed intake, feed conversion ratio, and mortality rate), and cecal histology. The current study demonstrates that combining liver free and chitosan has superior preventive and therapeutic benefits than either alone, and they could also be used as alternative anticoccidial agents.

Anti-Eimeria tenella activity of Ethanamizuril in vitro and in vivo.

The prevalence of chicken coccidiosis in the poultry industry is a significant concern, further exacerbated by the emergence of drug-resistant coccidia resulting from the indiscriminate use of medications. Ethanamizuril, a novel triazine anti-coccidial compound, has been used to combat drug resistance. Currently, it is known that Ethanamizuril acts on the second-generation merozoites and early gametogenesis stages of Eimeria. Limited information exists regarding its impact on the early merozoites and exogenous stage of Eimeria. In the present study, the anti-coccidial properties of Ethanamizuril were evaluated both in vitro and in vivo. The in vitro experiments demonstrated that Ethanamizuril effectively inhibits the sporulation of E. tenella oocysts in a dose-dependent manner and significantly reduces the sporozoite excystation rate. Furthermore, in vivo tests revealed that treatment with 10 mg/L Ethanamizuril in drinking water significantly decreased the copy number of first-generation and secondary-generation merozoites in the chicken cecum, indicating that it can inhibit the development of whole schizonts development. Moreover, treatment with Ethanamizuril demonstrated excellent protective efficacy with an anti-coccidial index (ACI) of 180.2, which was manifested through higher body weight gains, lighter cecal lesion, lower fecal oocyst shedding score and reduced liver index. Collectively, this study suggests that Ethanamizuril effectively treats E. tenella infection by inhibiting both endogenous and exogenous stages development.

Silymarin effectively prevents and treats Eimeria tenella infection in chicks.

Silymarin, a botanical medicine derived from milk thistle seeds and is known to improve chicken growth and gut health when added to the feed. However, its role in the prevention and treatment of chicken coccidiosis remains unclear. This study investigated the efficacy of various doses of silymarin in preventing and treating Eimeria tenella infection in chicks. A total of 180 one-day-old specific pathogen-free chicks were randomized into six groups of 30 chicks each, no treatment (NC group); E. tenella infection (CC group); diclazuril medication during d 14 to 21 and E. tenella infection (DC group); and three groups infected with E. tenella and administered low, medium, or high doses of silymarin during d 12 to 21. All groups except NC were infected with E. tenella on d 14, with indicators observed on d 21. The growth performance was higher in the silymarin treated groups than that in the CC group, and the oocyst count per gram of manure, blood stool, and cecal lesion scores decreased. The medium-dose silymarin group exhibited the best treatment effect. Additionally, the silymarin groups displayed improved histological, morphology, and intestinal barrier integrity. The amounts of proinflammatory factors and harmful bacteria in the cecum were also reduced. Additionally, the activity of serum and cecal antioxidant enzymes, as well as the abundance of beneficial gut microbiota, increased in the cecum. In conclusion, this study demonstrated that silymarin can prevent and treat E. tenella infections. These data provide a scientific and conceptual basis for the development of a botanical dietary supplement from silymarin for the treatment and control of coccidiosis in chicks.

Study on the effects of toosendanin against Eimeria tenella and its impact on the apoptosis of cecal cells in chicks.

To investigate the therapeutic effect of toosendanin (TSN) against Eimeria tenella (E. tenella) in chicks. In this experiment, a chick model of artificially induced E. tenella infection was established. The anti-coccidial effect was investigated by treating different doses of TSN. A preliminary mechanism of action was conducted, using cecal cell apoptosis as a starting point. TSN at the concentration of 5 mg/kg BW showed the best effect against E. tenella with the ACI value of 164.35. In addition, TSN reduced pathological damage to cecal tissue, increased the secretion of glycogen and mucus in cecal mucosa, and enhanced the mucosal protective effect. It also elevated the levels of IFN-γ, IL-2, and IgG in serum, and raised the sIgA content in cecal tissue of infected chicks, thereby improving overall immune function. TSN was observed to promote the apoptosis of cecum tissue cells by TUNEL staining analysis. Immunohistochemistry analysis revealed that in TSN-treated groups, the expression of Caspase-3 and Bax was elevated, while the expression of Bcl-2 was reduced. TSN induced apoptosis in host cells by dose-dependently decreasing the Bcl-2/Bax ratio and upregulating Caspase-3 expression. In summary, TSN exhibited significant anticoccidial efficacy by facilitating apoptosis in host cecal cells, with the most pronounced effect observed at a dosage of 5 mg/kg body weight.

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.

Prophylactic and Therapeutic Efficacy of Ultrasonicated Rosmarinus officinalis Ethanolic Extract and its Chitosan-Loaded Nanoparticles Against Eimeria tenella Infected Broiler Chickens.

PURPOSE: The in vivo efficacy of ultrasonicated Rosmarinus officinalis ethanolic extract (UROEE) and its chitosan-loaded nanoparticles (UROEE-CsNPs) was investigated as a dietary prophylactic agent and as a therapeutic treatment against Eimeria tenella infected broiler chickens. METHODS: Chickens were infected with 4 × 104 E. tenella oocysts at 21 days old for primary infection and with 8 × 104 oocysts at 35 days old for secondary infection. Eleven experimental groups were conducted. Dietary addition of 100 mg/kg UROEE and 20 mg/kg for CsNPs as well as UROEE-CsNPs were included for prophylactic groups from day 1 to 42. The same doses were used for therapeutic treatment groups for 5 constitutive days. Oocyst output in feces was counted. Histopathological and immunohistochemical studies were conducted. Gene expression of pro-inflammatory cytokines as IFN-γ, IL-1ß and IL-6 as well as anti-inflammatory cytokines as IL-10 and TGF-ß4 was analyzed using semi-quantitative reverse transcriptase-PCR. RESULTS: The results showed an efficacy of UROEE, CsNPs and UROEE-CsNPs in reduction of oocyst excretion and improving the cecal tissue architecture. CD4+ and CD8+ T lymphocytes protein expression were reduced. E. tenella infection lead to upregulation of pro-inflammatory cytokines as IFN-γ, IL-1ß, IL-6 and anti-inflammatory cytokines as TGF-ß4 following primary infection, while their expression was downregulated following secondary infection. CONCLUSION: The dietary prophylactic additives and therapeutic treatments with UROEE, CsNPs and UROEE-CsNPs could decrease the inflammatory response to E. tenella as indicated by oocyst output reduction, histopathological improvements, CD4+ and CD8+ T cells protein expression reduction as well as reducing mRNA expression levels of the tested cytokines following primary and secondary infections. Consequently, these results will help to develop better-combating strategies for the control and prevention of coccidiosis on poultry farms as a dietary prophylactic agent or as a therapeutic treatment.

Effects of host vimentin on Eimeria tenella sporozoite invasion.

BACKGROUND: Chicken coccidiosis is a parasitic disease caused by Eimeria of Apicomplexa, which has caused great economic loss to the poultry breeding industry. Host vimentin is a key protein in the process of infection of many pathogens. In an earlier phosphorylation proteomics study, we found that the phosphorylation level of host vimentin was significantly regulated after Eimeria tenella sporozoite infection. Therefore, we explored the role of host vimentin in the invasion of host cells by sporozoites. METHODS: Chicken vimentin protein was cloned and expressed. We used qPCR, western blotting, and indirect immunofluorescence to detect levels of mRNA transcription, translation, and phosphorylation, and changes in the distribution of vimentin after E. tenella sporozoite infection. The sporozoite invasion rate in DF-1 cells treated with vimentin polyclonal antibody or with small interfering RNA (siRNA), which downregulated vimentin expression, was assessed by an in vitro invasion test. RESULTS: The results showed that vimentin transcription and translation levels increased continually at 6-72 h after E. tenella sporozoite infection, and the total phosphorylation levels of vimentin also changed. About 24 h after sporozoite infection, vimentin accumulated around sporozoites in DF-1 cells. Treating DF-1 cells with vimentin polyclonal antibody or downregulating vimentin expression by siRNA significantly improved the invasion efficiency of sporozoites. CONCLUSION: In this study, we showed that vimentin played an inhibitory role during the invasion of sporozoites. These data provided a foundation for clarifying the relationship between Eimeria and the host.

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.

Anticoccidial effect of Origanum majoranum aqueous extract on Eimeria tenella-infected chicken.

Avian coccidiosis, an important protozoal disease of chicken triggered by coccidian protozoa of genus Eimeria, causes considerable economic losses to broiler producers. The study was designed to assess the efficiency of Origanum majoranum aqueous extract (OMAE) on E. tenella-infected broiler chicken. Birds were divided into four groups including: positive control (PC, challenged with 5×104 sporulated oocysts of E. tenella at the 12th day of age), PC+OMAE (challenged with E. tenella oocysts at the 12th day of age and received OMAE (125 mg/kg BW) orally, started at the 7th day of age, and continued for 14 consecutive days), OMAE (received OMAE (125 mg/kg BW) orally, at the 7th day of age, for 14 consecutive days), and negative control (received basal diet only). Anticoccidial efficacy of OMAE was evaluated by complete blood picture, serum chemistry, serum protein electrophoresis, antioxidants markers, cecal oocysts count, and cecal lesions score. Briefly, collected data indicated that supplementation of OMAE could increase antioxidants concentrations and improve changes in hematobiochemical parameters and serum protein fractions, as well as decrease cecal oocysts count and reduce cecal lesion scores in E. tenella-infected birds. In conclusion, OMAE restores oxidant-antioxidant balance, and its supplementation in broiler chicken can alleviate E. tenella-infection and reduce its severity.

Exogenous nitric oxide stimulates early egress of Eimeria tenella sporozoites from primary chicken kidney cells in vitro.

Egress plays a vital role in the life cycle of apicomplexan parasites including Eimeria tenella, which has been attracting attention from various research groups. Many recent studies have focused on early egress induced by immune molecules to develop a new method of apicomplexan parasite elimination. In this study, we investigated whether nitric oxide (NO), an immune molecule produced by different types of cells in response to cytokine stimulation, could induce early egress of eimerian sporozoites in vitro. Eimeria tenella sporozoites were extracted and cultured in primary chicken kidney cells. The number of sporozoites egressed from infected cells was analyzed by flow cytometry after treatment with NO released by sodium nitroferricyanide (II) dihydrate. The results showed that exogenous NO stimulated the rapid egress of E. tenella sporozoites from primary chicken kidney cells before replication of the parasite. We also found that egress was dependent on intra-parasitic calcium ion (Ca2+) levels and no damage occurred to host cells after egress. The virulence of egressed sporozoites was significantly lower than that of fresh sporozoites. The results of this study contribute to a novel field examining the interactions between apicomplexan parasites and their host cells, as well as that of the clearance of intracellular pathogens by the host immune system.

TITLE: L'oxyde nitrique exogène stimule in vitro la sortie précoce des sporozoïtes d'Eimeria tenella des cellules primaires de rein de poulet. ABSTRACT: La sortie des cellules joue un rôle vital dans le cycle de vie des parasites Apicomplexa, y compris Eimeria tenella, ce qui a attiré l'attention de plusieurs groupes de recherche. De nombreuses études récentes se sont concentrées sur la sortie précoce induite par des molécules immunitaires, pour développer une nouvelle méthode d'élimination des parasites Apicomplexa. Dans cette étude, nous avons examiné si l'oxyde nitrique (NO), une molécule immunitaire produite par différents types de cellules en réponse à la stimulation des cytokines, pouvait induire in vitro une sortie précoce des sporozoïtes des Eimeria. Les sporozoïtes d'E. tenella ont été extraits et cultivés dans des cellules primaires de rein de poulet. Le nombre de sporozoïtes sortant des cellules infectées a été analysé par cytométrie en flux après traitement avec du NO libéré par le nitroferricyanure de sodium (II) dihydraté. Les résultats ont montré que le NO exogène stimulait la sortie rapide des sporozoïtes d'E. tenella des cellules primaires de rein de poulet avant la réplication du parasite. Nous avons également constaté que la sortie dépendait des niveaux intra-parasitaires d'ions calcium (Ca2+) et qu'aucun dommage n'est survenu aux cellules hôtes après la sortie. La virulence des sporozoïtes sortis était significativement inférieure à celle des sporozoïtes frais. Les résultats de cette étude contribuent à un nouveau domaine d'étude des interactions entre les parasites Apicomplexa et leurs cellules hôtes, ainsi qu'à celui relatif à l'élimination des pathogènes intracellulaires par le système immunitaire de l'hôte.

Effects of probiotic and Bidens pilosa on the performance and gut health of chicken during induced Eimeria tenella infection.

AIM: In this study, we have examined the individual and combined protective mechanism of probiotic and Bidens pilosa on the performance and gut health of chickens during Eimeria tenella infection over a 29-day experimental trial. METHODS AND RESULTS: A total of one hundred and fifty 1-day-old chickens were equally distributed into five treatment groups with three biological replicates: two groups were allocated as control groups (control group untreated unchallenged, CG and control positive untreated challenged, CPG) and three groups were fed diets with probiotic (PG), B. pilosa (BPG) and probiotic + B. pilosa (PG + BPG) and challenged with E. tenella. Birds of all groups were assessed for pre and post-infection body weights, oocysts shedding, caecal lesion scores and mRNA expression levels of apoptosis related proteins (Bcl-2, Bax and caspase-3), antioxidant enzymes (CAT and SOD 1), pro-inflammatory cytokines (IL-6 and IL-8) and tight junction proteins (CLDN 1 and ZO 1). Our results revealed that during infection (day 21-29), E. tenella challenged chickens significantly decreased the body weight compared with uninfected control chickens; however, there was no significant effect on body weight of chickens fed with probiotic, B. pilosa and probiotic + B. pilosa was observed. Eimeria tenella challenged untreated birds increased (P < 0·05) oocysts shedding, destructive ratio of caeca and mortality as compared to treated challenged birds. CPG group up-regulated the mRNA expression levels of anti-apoptosis protein Bcl-2 while down-regulated the pro-apoptosis protein Bax relative to PG, BPG and PG + BPG groups. Moreover chickens fed probiotic, B. pilosa and probiotic + B. pilosa diets enhanced the activities of antioxidant enzymes, pro-inflammatory cytokines and tight junction proteins with the comparison of control positive untreated challenged chickens. CONCLUSION: These findings elaborated that feed supplementation of probiotic and B. pilosa (individually or in combination) appeared to be effective in inhibiting the occurrence of disease and decreasing the severity of Eimeria infection in chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: This study explained the underlying anti-coccidial mechanism in which probiotic and B. pilosa (individually and/or in combination) improve the performance of chicken and protect against gut inflammatory responses caused by E. tenella.

Effects of diclazuril on the expression of enolase in second-generation merozoites of Eimeria tenella.

Eimeria tenella is an obligate intracellular parasite of the chicken cecum; it brings huge economic loss to the chicken industry. Enolase is a multifunctional glycolytic enzyme involved in many processes of parasites, such as infection and migration. In this study, the effect of diclazuril on the expression of enolase in second-generation merozoites of E. tenella (EtENO) was reported. The prokaryotic expression plasmid pET-28a-EtENO was constructed and transformed into Escherichia coli BL21 (DE3). Then, it was subjected to expression under the induction of isopropyl-ß-D-1-thiogalactopyranoside. The expressed products were identified and purified. The purified EtENO protein was used for antibody preparation. The EtENO mRNA and protein expression levels were analyzed via real-time PCR and Western blotting. Localization of EtENO on the merozoites was examined by immunofluorescence technique. The mRNA and protein expression levels of EtENO were decreased by 36.3 and 40.36%, respectively, by diclazuril treatment. EtENO distributed in the surface, cytoplasm, and nucleus of the infected/control group. With diclazuril treatment, it was significantly reduced in the surface and cytoplasm and even disappeared in the nucleus of the infected/diclazuril group. These observations suggested that EtENO may play an important role in mechanism of diclazuril anticoccidial action and be a potential drug target for the intervention with E. tenella infection.

Vaccination with transgenic Eimeria tenella expressing Eimeria maxima AMA1 and IMP1 confers partial protection against high-level E. maxima challenge in a broiler model of coccidiosis.

BACKGROUND: Poultry coccidiosis is a parasitic enteric disease with a highly negative impact on chicken production. In-feed chemoprophylaxis remains the primary method of control, but the increasing ineffectiveness of anticoccidial drugs, and potential future restrictions on their use has encouraged the use of commercial live vaccines. Availability of such formulations is constrained by their production, which relies on the use of live chickens. Several experimental approaches have been taken to explore ways to reduce the complexity and cost of current anticoccidial vaccines including the use of live vectors expressing relevant Eimeria proteins. We and others have shown that vaccination with transgenic Eimeria tenella parasites expressing Eimeria maxima Apical Membrane Antigen-1 or Immune Mapped Protein-1 (EmAMA1 and EmIMP1) partially reduces parasite replication after challenge with a low dose of E. maxima oocysts. In the present study, we have reassessed the efficacy of these experimental vaccines using commercial birds reared at high stocking densities and challenged with both low and high doses of E. maxima to evaluate how well they protect chickens against the negative impacts of disease on production parameters. METHODS: Populations of E. tenella parasites expressing EmAMA1 and EmIMP1 were obtained by nucleofection and propagated in chickens. Cobb500 broilers were immunised with increasing doses of transgenic oocysts and challenged two weeks later with E. maxima to quantify the effect of vaccination on parasite replication, local IFN-γ and IL-10 responses (300 oocysts), as well as impacts on intestinal lesions and body weight gain (10,000 oocysts). RESULTS: Vaccination of chickens with E. tenella expressing EmAMA1, or admixtures of E. tenella expressing EmAMA1 or EmIMP1, was safe and induced partial protection against challenge as measured by E. maxima replication and severity of pathology. Higher levels of protection were observed when both antigens were delivered and was associated with a partial modification of local immune responses against E. maxima, which we hypothesise resulted in more rapid immune recognition of the challenge parasites. CONCLUSIONS: This study offers prospects for future development of multivalent anticoccidial vaccines for commercial chickens. Efforts should now be focused on the discovery of additional antigens for incorporation into such vaccines.