Oocyst Output Research Articles

Holothuria polii Extract as a Potential Anticoccidial Agent: Evidence of Its MUC2 Regulatory Impact in Murine Jejunum.

Eimeria is a parasite that causes coccidiosis in a variety of animals, leading to nutritional imbalances, issues with food digestion and absorption, low weight, and intestinal inflammation of varying degrees in its hosts. Anticoccidial medication resistance has become a significant obstacle to disease control efforts, leading to a renewed focus on discovering novel chemicals for alternative therapeutic approaches. The purpose of this study was to determine the efficacy of Holothuria polii extract (HpE) in treating eimeriosis in mice. Five groups of mice were studied, with E. papillata sporulated oocysts (1 × 103) used to infect three groups experimentally. The first group was treated with 200 mg/kg of HpE, the second group with Amprolium (120 mg/kg), and the third group was left untreated as a control. The fourth and fifth groups were uninfected, with one of them treated with 200 mg/kg of HpE and the other used as a negative control. The results revealed that HpE has 10 phytochemical compounds according to GC-MS analysis. Treatment with HpE resulted in a substantial decrease in oocyst output in feces. Also, the parasitic stages showed morphometric alterations, with reductions in the measurements compared with the infected, untreated mice. Moreover, expression of the mucin gene (MUC2) was higher in treated mice compared to infected ones, with significant increases in goblet cell numbers, which provided additional evidence for the activity of HpE as an anticoccidial product. In conclusion, there are a wide variety of natural products and many have been employed in folk medicine for treating a range of parasitic diseases.

Eimeria tenella rhoptry neck protein 2 plays a key role in the process of invading the host intestinal epithelium

The Apicomplexa parasitic phylum rhoptry neck protein 2 (RON2) plays a key role in the process of invading host cells. Eimeria tenella, an intracellular protozoan shares a similar conserved invasion pattern. However, whether E. tenella RON2 participates in the process of invading the host intestinal epithelium is poorly understood. In this study, the sequence of EtRON2 was analyzed and expressed. The expression of the truncated extracellular N-terminal fragment of EtRON2 (403–700 aa, designated EtRON2403–700) with a molecular mass of 38.3 kDa. EtRON2 in the sporozoite protein was detected at 151.4 kDa by rabbit anti-rEtRON2403–700 antibody. Immunofluorescence results showed that EtRON2 was mainly localized to the nucleus and apex of the E. tenella sporozoite. qPCR results showed that the highest expression level of EtRON2 was detected in sporulated oocysts compared with other developmental stages of E. tenella. In vitro invasion inhibition assays showed that the capacity of sporozoites to invade DF-1 cells was significantly inhibited after pretreatment with the rabbit anti-rEtRON2403–700 antibody. Silencing the EtRON2 gene by RNA interference (RNAi) significantly inhibited EtRON2 expression and significantly reduced the invasion of DF-1 cells by sporozoites. In vivo experiments revealed a significant decrease parasite burden and oocyst outputs in chicks after infection with EtRON2 gene-silenced sporozoites by cloacal inoculation. Recombinant EtRON2403–700 (rEtRON2403–700) immunizes chicks effectively against E. tenella infection by inducing humoral immunity and upregulating IFN-γ and CD8+ T lymphocytes. Furthermore, chicks exhibited increased relative weight gain rates, lower cecum lesion scores, and reduced oocyst outputs during the E. tenella challenge. H&E staining showed that the cecum tissue of chicks immunized with rEtRON2403–700 showed relatively mild histopathological changes. In conclusion, the results of this study demonstrated that EtRON2 plays a key role in E. tenella invasion of the host intestinal epithelium and provides a potential target for vaccines against E. tenella infection.

Anticoccidial and Antioxidant Activities of an Ethanolic Extract of Teucrium polium Leaves on Eimeria papillate-Infected Mice.

Eimeria spp. are responsible for the economic loss of both domestic and wild animals due to coccidiosis, the most common parasitic disease. The resistance to currently available drugs used to treat coccidiosis has been proven. Medicinal plants that contain physiologically active phytochemicals have been widely used in traditional medicine. Teucrium polium leaf extract (TPLE) has been shown to exhibit pharmacological, antioxidant, and anticoccidial properties in different experiments. Here, our investigation focused on how T. polium leaf extract affected the way that Eimeria papillate caused intestinal injury in mice. Thirty-five male Swiss albino mice were divided into seven groups, as follows: group I: untreated and uninfected (negative control); group II: uninfected, treated group with TPLE (150 mg/kg b.w); and group III: infected untreated (positive control). Groups III-VII were orally administered 103 sporulated E. papillata oocysts. A total of 60 min after infection, groups IV-VI were treated for five successive days with 50, 150, and 250 mg/kg b.w TPLE, respectively, while group VII was treated with amprolium (120 mg/kg b.w.). The mice had been euthanized on the fifth day post-infection, and the jejunum tissues were prepared for histology and oxidative stress studies. A total of 150 mg/kg of TPLE was the most effective dosage, significantly decreasing oocyst output by about 80.5%, accompanied by a significant reduction in the number of developmental parasitic phases in jejunal sections. In addition, the decrease in the number of goblet cells in the jejuna of mice raised after treatment. Also, TPLE greatly diminished the body weight loss of infected mice. Moreover, our research proved that TPLE reduced oxidative damage due to E. papillata infection via decreasing intestinal malondialdehyde (MDA) and nitric oxide (NO) levels and increasing reduced superoxide dismutase (SOD) and glutathione (GSH) levels. These results demonstrated that TPLE had potent anticoccidial properties. TPE's efficacy as a natural antioxidant has also been demonstrated in reducing oxidative stress and enhancing antioxidant systems to mitigate biochemical and histological changes in the jejunum caused by E. papillata.

Study on the alleviative effect of Lactobacillus plantarum on Eimeria falciformis infection.

Coccidia of the genus Eimeria are specialized intracellular parasitic protozoa that cause severe coccidiosis when they infect their hosts. Animals infected with Eimeria develop clinical symptoms, such as anorexia, diarrhea, and hematochezia, which can even cause death. Although the current preferred regimen for the treatment of coccidiosis is antibiotics, this treatment strategy is limited by the ban on antibiotics and the growing problem of drug resistance. Therefore, the exploration of alternative methods for controlling coccidiosis has attracted much attention. Lactobacillus plantarum has been shown to have many beneficial effects. In this study, L. plantarum M2 was used as a research object to investigate the effect of L. plantarum on intestinal inflammation induced by infection with Eimeria falciformis in mice by detecting indicators, such as oocyst output, serum cytokines, and the intestinal microbiota. Compared with that in the infection group, the percent weight loss of the mice that were administered with L. plantarum M2 was significantly reduced (P < 0.05). Supplemented L. plantarum M2 and probiotics combined with diclazuril can reduce the total oocyst output significantly (P < 0.05, P < 0.001). L. plantarum M2 had outstanding performance in maintaining intestinal barrier function, and the levels of the mucin MUC1 and the tight junction protein E-cadherin were significantly elevated (P < 0.01, P < 0.05). Studies have shown that probiotic supplementation can alleviate adverse reactions after infection and significantly improve intestinal barrier function. In addition, probiotics combined with diclazuril could optimize the partial efficacy of diclazuril, which not only enhanced the effect of antibiotics but also alleviated their adverse effects. This study expands the application of probiotics, provides new ideas for alternative strategies for coccidia control, and suggests a basis for related research on lactobacilli antagonizing intracellular pathogen infection.IMPORTANCECoccidia of the genus Eimeria are specialized intracellular parasitic protozoa, and the current preferred regimen for the treatment of coccidiosis is antibiotics. However, due to antibiotic bans and drug resistance, the exploration of alternative methods for controlling coccidiosis has attracted much attention. In this work, we focused on Lactobacillus plantarum M2 and found that probiotic supplementation can alleviate adverse reactions after infection and improve intestinal barrier function. This study proposes the possibility of using lactic acid bacteria to control coccidiosis, and its potential mechanism needs further exploration.

Trifolium pratense as a novel phytogenic supplement, is an anticoccidial agent in chickens

Coccidiosis, caused by a protozoan parasite of the genus Eimeria, is one of the most severe contagious parasite diseases affecting the poultry industry worldwide. Using phytogenics to prevent chicken coccidiosis is a strategy aimed at combating the increasing issue of drug-resistant strains of Eimeria spp. This study demonstrates the anticoccidial activities of a medicinal herb, Trifolium pratense (TP) powder, and its ethanolic extract (designated TPE) against Eimeria spp. TPE exhibited significant suppressive activity against E. maxima oocyst sporulation and E. tenella sporozoite invasion and reproduction in Madin-Darby bovine kidney cells. Furthermore, administration of basal chicken diets containing TP powder or TPE to Eimeria-infected chickens significantly reduced the output of oocysts and severity of intestinal lesions. Dietary supplementation with TP significantly improved relative weight gain in E. tenella- and E. acervulina-infected chickens, while there was no significant improvement in E. maxima-infected chickens. The anticoccidial activities of TP and TPE on E. acervulina, E. tenella and E. maxima were further supported by anticoccidial index scores, which showed greater efficacy than those of amprolium, a commercial coccidiostat used in poultry. TP supplementation positively impacted the primary metabolism of chickens challenged with E. tenella or E. acervulina. The chemical fingerprints of TPE were established using liquid column chromatography; TPE contained 4 major compounds: ononin, sissotrin, formononetin, and biochanin A. In addition, various spectrometric methods were used to ensure the batch-to-batch consistency of TP/TPE. In conclusion, T. pratense is demonstrated to be a novel phytogenic supplement that can be used to control Eimeria-induced coccidiosis in chickens.

Cassia alata's dual role in modulating MUC2 expression in Eimeria papillata-infected jejunum and assessing its anti-inflammatory effects.

Coccidiosis poses significant hazards to animals, particularly in terms of compromised health, reduced productivity, and economic losses in livestock farming. The conventional treatments for coccidiosis often involve synthetic drugs, contributing to concerns about drug resistance and environmental impact. The pressing need for eco-friendly alternatives is highlighted in this study, emphasizing the importance of exploring medicinal plants like Cassia alata leaf extracts (CAE) against Eimeria papillata-induced infection in mice. The CAE exhibited significant phenolic (2.17 ± 0.03 g/100 g) and flavonoid (0.14 ± 0.01 g/100 g) content and demonstrated notable antioxidant activity. In infected mice, the CAE treatment led to a substantial reduction in oocyst output (~6 fold), ameliorating necrotic enteritis and inflammatory changes in the jejunum. Additionally, CAE treatment increased goblet cell numbers (9.3 ± 0.1 / villus) and decreased macrophage infiltration in the intestinal villi. Molecular analyses revealed CAE's positive modulation of MUC2 gene and notably reduced the levels of pro-inflammatory cytokines (specifically IL-1β, IL-10, and IFN-γ) when contrasted with the infected cohort. Furthermore, CAE treatment significantly reduced nitric oxide levels (44.03 ± 2.4 μmol/mg), showcasing its anti-inflammatory properties. The findings of this study not only contribute to the understanding of CAE's therapeutic potential but also underscore the importance of seeking eco-friendly alternatives in the face of coccidiosis challenges, addressing both the well-being of animals and the sustainability of agricultural practices. RESEARCH HIGHLIGHTS: Cassia alata extract (CAE) exhibited significant phenolic and flavonoid content, displaying notable antioxidant activity. In infected mice, CAE treatment led to a substantial reduction in oocyst output, ameliorating necrotic enteritis and inflammatory changes in the jejunum. CAE treatment increased goblet cell numbers and decreased macrophage infiltration in the intestinal villi, while molecular analyses revealed its positive modulation of the MUC2 gene and notable reduction in pro-inflammatory cytokine levels. Additionally, CAE treatment significantly reduced nitric oxide levels, showcasing its anti-inflammatory properties.

Effect of dietary treatments on performance, oocysts shedding and lesion scores in broiler chickens experimentally challenged with Eimeria infection

Although anticoccidials effectively control coccidiosis, a needed reduction in the reliance on antimicrobials in animal production leads to the exploration of alternative compounds. The present study aimed to test five different dietary treatments to counteract the negative impact of coccidiosis on broiler chickens’ health and performance. 1-day-old male Ross 308 broilers (n = 960) were randomly assigned to one of eight treatments, with six cages per treatment (20 birds/cage). To the diet of the broiler chickens of treatments (Trt) 1–5, a synbiotic was added from d0-10. From d10–28, birds of Trt1 and Trt2 were fed synbiotics, whereas birds of Trt3 were fed diets with glutamine, and birds of Trt4 and Trt5 were fed diets with a combination of β-glucans and betaine. From d28-35 onwards, birds of Trt1 were fed a diet with a synbiotic, whereas birds of Trt2-4 received diets with glutamine, and birds of Trt5 were fed a non-supplemented diet. Birds of the positive control group (PC; Trt6) were fed a standard diet supplemented with an anticoccidial (Decoquinate). The challenged negative control (NCchall; Trt7) and non-challenged negative control (NC) Trt8 were fed a standard diet without anticoccidial or other dietary treatment. At 7 days (d) of age, all birds were inoculated with 1 023, 115, and 512 sporulated oocysts of E. acervulina, E. maxima, and E. tenella, respectively, except for Trt8. Body weight gain (BWG), feed intake, and feed conversion ratio were assessed for each feeding phase (d0-10, d10-28 and d28-35) and overall experimental period (d0-35). Oocyst shedding, Eimeria lesion scores, cecal length, and relative weight were assessed at d13, d22, d28 and d35. Additionally, oocyst shedding was determined at d9 and d17. Litter quality was evaluated at d27 and d34, and footpad lesions at d34. During the starter (d0-10) and finisher (d28-35) periods, performance did not differ between the treatments. During the grower period (d10-28), Trt6 (PC) and Trt8 (NC) chickens had the highest BWG of all treatments (P < 0.001). Dietary treatment had no effect on litter quality and severity of footpad lesions. In the PC group (Trt6), low oocyst excretion and lesion scores were found. When comparing Trt1-5 with NCchall (Trt7), none of the treatments significantly reduced oocyst output or lesion scores. In conclusion, in this experiment, none of the dietary treatments performed similar or better compared to the PC group (Trt6) regarding performance or reducing Eimeria oocyst shedding or lesion scores.

Susceptibility and cecal microbiota alteration to Eimeria-infection in Yellow-feathered broilers, Arbor Acres broilers and Lohmann pink layers

Coccidiosis, which is caused by Eimeria species, results in huge economic losses to the poultry industry. Arbor Acres (AA) broilers and yellow-feathered broilers are the dominant broilers in northern and southern China, respectively. However, their susceptibility to coccidiosis has not been fully compared. In this study, the susceptibility of yellow-feathered broilers, AA broilers and Lohmann pink layers to E. tenella was evaluated based on mortality rate, relative body weight gain rate, intestinal lesion score, oocyst output, anticoccidial index (ACI), and cecum weight and length. The yellow-feathered broilers were shown to produce significantly fewer oocysts with higher intestinal lesion score compared to AA broilers, which had the highest growth rates and ACI scores. Subsequently, changes in the cecal microbiota of the 3 chicken lines before and after high-dose infection (1 × 104 oocysts) with E. tenella were determined by 16S rRNA sequencing. The results showed that composition of the microbiota changed dramatically after infection. The abundance of Firmicutes and Bacteroidetes in the infected chickens decreased, and Proteobacteria increased significantly among the different chicken lines. At the genus level, Escherichia increased significantly in all 3 groups of infected chickens, but Lactobacillus decreased to 0% in the infected yellow-feathered broilers. The results of the study indicate that the susceptibility to E. tenella varies among the 3 chicken lines, and that changes in intestinal microbiota by E. tenella-infection among the different chicken lines had a similar trend, but to different degrees. This study provides basic knowledge of the susceptibility in the 3 chicken lines, which can be helpful for the control and prevention of coccidiosis.

Effects of three kinds of Chinese herbs on growth performance, oocysts output and gut microbiota in growing lambs with coccidiosis.

Coccidiosis is a protozoan intestinal disease that reduces the production of the sheep industry and causes large economic losses for sheep. Although chemically synthesised drugs are routinely employed to treat coccidiosis in sheep, the anticoccidial drug resistance and drug residues in edible meat have prompted an urgent search for alternatives. Herein, the anticoccidial properties of diclazuril, a conventional anticoccidial drug, and Allium sativum, Houttuynia cordata and Portulaca oleracea were assessed. Forty 45-day-old lambs naturally infected with Eimeria spp. were selected and randomly divided into five groups. The results showed that the sheep treated for coccidiosis had considerably decreased average daily gain (ADG) during both administration and withdrawal of the drug compared to the control group. Furthermore, at days 14, 21, 28 and 35, respectively, the three herbs and diclazuril had similar anticoccidial effects, with lower oocysts per gram (OPG) than the control group. On day 78, OPG in the three herbal groups was significantly lower than in the diclazuril group. In addition, the abundance and composition of the gut microbiota were changed in sheep treated with the three herbs and diclazuril compared to the untreated sheep. Moreover, some intestinal microorganisms have a correlation with OPG and ADG when using Spearman correlation analysis. In summary, our results suggest that all three herbs produce anticoccidial effects similar to diclazuril and modulate the balance of gut microbiota in growing lambs.

Effects of Six Natural Compounds and Their Derivatives on the Control of Coccidiosis in Chickens.

Chicken coccidiosis costs the poultry industry over GBP 10 billion per year. The main method of preventing and controlling coccidiosis in chickens continues to be the use of drugs. Unfortunately, the prevalence of drug resistance in the field reduces or even eliminates the effectiveness of drugs, and drug residues in the food supply chain can also can be harmful to humans. Therefore, safe and effective anticoccidial drugs are urgently needed. Natural products have many advantages such as being safe, effective and inexpensive and are a sustainable way to control coccidiosis. In this study, the anticoccidial effects of six natural compounds were tested by Eimeria tenella infection. Oocyst production, cecum lesion, body weight gain, feed conversion ratio, and intestinal microbiota were measured. The results showed that nerolidol had a moderate effect on maintaining both body weight gain and feed conversion ratio. Silymarin and dihydroartemisinin showed significant anticoccidial effects by reducing total oocyst output. Dihydroartemisinin also significantly reduced the cecum lesion caused by Eimeria infection, but this compound may be toxic to the host at such informed doses because it decreases growth and survival rates. In addition, both silymarin and dihydroartemisinin partly restored the microbiota after challenge. This indicates that silymarin, dihydroartemisinin, and nerolidol are effective in the control of chicken coccidiosis. Our data provide basic knowledge about the anticoccidial effects of such natural compounds/derivates.

Fecal microbiota impacts development of Cryptosporidium parvum in the mouse

The dependence of Cryptosporidium parasites on host cell metabolites suggests that the development of nutritional interventions to limit parasite proliferation should be feasible. Based on this concept, we are testing dietary interventions to affect the enterocytes’ metabolism in a manner that limits intracellular multiplication of the parasite. We hypothesize that changes in the metabolic pathways encoded by the gastro-intestinal tract microbiota may restrict parasite proliferation. To identify taxonomic and metabolic features of the microbiota associated with severity of cryptosporidiosis, as determined by estimating oocyst output, we characterized the fecal microbiota from mice experimentally infected with Cryptosporidium parvum. To eliminate the confounding effect of the interaction between co-housed mice, as well as facilitate the identification of microbiota markers associated with severity of cryptosporidiosis, fecal microbiota from individually caged mice were analyzed. Variation partitioning analysis applied to 16S sequence data from 25 mice belonging to four experiments shows that experiment was by far the biggest source of microbiota variation. Severity of cryptosporidiosis explained a smaller, though significant, fraction of microbiota variation. Notably, this effect was significant in the pre-patent phase of the infection, before mice excreted oocysts. These results are consistent with the pre-patent intestinal microbiota having a modest, but measurable, effect on cryptosporidiosis.

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

PurposeThe 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.MethodsChickens were infected with 4 × 104E. 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-1β and IL-6 as well as anti-inflammatory cytokines as IL-10 and TGF-β4 was analyzed using semi-quantitative reverse transcriptase-PCR.ResultsThe 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-1β, IL-6 and anti-inflammatory cytokines as TGF-β4 following primary infection, while their expression was downregulated following secondary infection.ConclusionThe 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.Graphical

Anticoccidial and jejunum-protective effects of Krameria lappacea roots extract on experimental Eimeria papillata infection.

Coccidiosis is a protozoan parasitic disease caused by Eimeria species and affects wild and domestic animals. Coccidiostats are currently available to control this disease, although drug resistance has been confirmed for all of them. As a result, there is an urgent need to identify eco-friendly agents to control and treat this disease. This study aimed to investigate the ameliorative role of the Krameria lappacea roots extract (KLRE) on the outcome of coccidiosis induced by Eimeria papillata. Male C57BL/6 mice were divided into seven groups (5 mice/group), as follows: Group 1: noninfected-nontreated (control group), Group 2: noninfected-treated group with KLRE (200 mg/kg), Group 3: infected-nontreated group, Group 4: infected-treated group with KLRE (50 mg/kg), Group 5: infected-treated group with KLRE (100 mg/kg), Group 6: infected-treated group with KLRE (200 mg/kg), and Group 7: infected-treated group with amprolium (120 mg/kg). Groups (3-7) were inoculated orally with 1 × 103 sporulated E. papillata oocysts. One hour after infection, groups (4-6) were daily treated for 5 days with KLRE and amprolium. On day 5 postinfection, oocyst output was determined, and mice were euthanized for the collection of jejuna then preparation of histological sections and jejunal homogenate was used for the determination of biochemical and oxidative damage markers. The coccidial infection induced weight loss of mice by 3.971%, which improved after KLRE to -1.512%. After KLRE treatment, the rate of feed intake was improved to be 52.21 ± 2.30 than those in infected group (40.47 ± 2.25). Oocyst output was significantly reduced in mice treated with KLRE (1.308 × 106 oocysts/g.feces) compared with those in the infected group (5.387 × 106 oocysts/g.feces). E. papillata infection induced marked histological alterations within jejunum tissue. After treatment, KLRE was able to impair the development of parasite stages (meronts, gamonts, and developing oocysts) in the jejunum through a significant reduction of number and size in comparison with the infected group. Infection with E. papillata induced a disturbance in the nutrient absorption in the jejunal mice tissue, which improved after the treatment with KLRE and amprolium. Also, KLRE counteracted significantly the E. papillata-induced loss of reduced glutathione and total antioxidant capacity. Our findings indicate that KLRE could be used as an alternative to the available coccidiostats currently available. RESEARCH HIGHLIGHTS: Krameria lappacea exhibit significant anticoccidial and antioxidant activities induced by E. papillata infection. Krameria lappacea exhibit significant improvement in the pathological alterations of the jejunal tissue induced by E. papillata infection.

Host protein EPCAM interacting with EtMIC8-EGF is essential for attachment and invasion of Eimeria tenella in chickens

The five epidermal growth factor-like domains (EGF) of Eimeria tenella microneme protein 8 (EtMIC8) (EtMIC8-EGF) plays a vital role in host cell attachment and invasion. These processes require interactions between parasite proteins and receptors on the surface of host cells. In this study, five chicken membrane proteins potentially interacting with EtMIC8-EGF were identified using the GST pull-down assay and mass spectrometry analysis, and only chicken (Gallus gallus) epithelial cell adhesion molecule (EPCAM) could bind to EtMIC8-EGF. EPCAM-specific antibody and recombinant EPCAM protein (rEPCAM) inhibited the EtMIC8-EGF binding to host cells in a concentration-dependent manner. Furthermore, the rEPCAM protein showed a binding activity to sporozoites in vitro, and a significant reduction of E. tenella invasion in DF-1 cells was further observed after pre-incubation of sporozoites with rEPCAM. The specific anti-EPCAM antibody further significantly decreased weight loss, lesion score and oocyst output during E. tenella infection, displaying partial inhibition of E. tenella infection. These results indicate that chicken EPCAM is an important EtMIC8-interacting host protein involved in E. tenella-host cell adhesion and invasion. The findings will contribute to a better understanding of the role of adhesion-associated microneme proteins in E. tenella.

A Study of Cross-Protection between Eimeria maxima Immunovariants.

For reasons unknown, Eimeria maxima is unique among Eimeria species infecting chickens in the immunovariability it displays among isolates from different geographical areas. Eimeria maxima oocysts (named EmaxAPU3) were isolated late in grow-out (6 weeks) from litter in a commercial broiler operation that was using Eimeria vaccination as the coccidiosis control program. Cross-protection studies (n = 4) were conducted in immunologically naïve chickens between EmaxAPU3 and two E. maxima lab strains (EmaxAPU1, EmaxAPU2) by immunizing with one E. maxima strain and challenging with either the homologous or heterologous E. maxima. As measured by oocyst output, immunization with EmaxAPU1 protected against homologous challenge (EmaxAPU1) and against heterologous challenge with EmaxAPU3, but not against EmaxAPU2. Similarly, immunization with EmaxAPU3 protected against homologous challenge (EmaxAPU3) and against heterologous challenge with EmaxAPU1, but not against EmaxAPU2. Immunization of chickens with EmaxAPU2 elicited a protective response against homologous challenge (EmaxAPU2), but not against EmaxAPU1 nor EmaxAPU3. The most plausible explanation for the appearance of this immunovariant late in grow-out is that E. maxima APU3 escaped immunity directed to E. maxima antigenic types in the commercial vaccine.

Fighting eimeriosis by using the anti-eimerial and anti-apoptotic properties of rhatany root extract.

Over the last decade, extensive use of coccidiostats to treat and control Eimeria infection has developed drug resistance, prompting the search for new alternative therapies. Rhatany is proven to have various pharmacological properties. The present study aimed to in vitro and in vivo evaluate the effect of Rhatany roots extract (RRE) as an anti-eimerial and anti-apoptotic agent against murine eimeriosis induced by Eimeria papillata. Phytochemical screening by gas chromatography-mass spectrometry analysis (GC-MS) was used to detect active compounds in RRE. In vitro anti-eimerial activity of RRE (200, 100, 50 mg/ml), amprolium, phenol, Dettol™, and formalin were studied after incubation with non-sporulated Eimeria oocysts. For the in vivo study, twenty-five male C57BL/6 mice were randomly allocated into five groups. Animals in the first group were just given distilled H2O, while those in the second group were given 200 mg/kg RRE for 5 days. The Eimeria parasite's oocysts were infected into the third, fourth, and fifth groups. For treatment, RRE (200 mg/kg) and amprolium (120 mg/kg) were orally given to the 4th and 5th groups for five days, respectively. All mice were euthanized, on day 5 post-infection, to collect the jejunal tissues under study. Investigations were undertaken into the oocyst output in feces and goblet cells in mice jejuna. Assays for glutathione peroxidase (GPx), hydrogen peroxide (H2O2), and myeloperoxidase (MPO) were also performed. In jejunal tissue, cysteine aspartic acid protease-3 (Caspase-3) was counted using immunohistochemistry, while BCL2-associated X protein (Bax) and B-cell lymphoma-2 (BCL-2) were assayed using ELISA. In addition, mRNA expression of the goblet cell response gene (MUC2) was detected using real-time PCR. Phytochemical screening by GC-MS demonstrated the presence of 22 compounds in the RRE. The in vitro study revealed that RRE significantly inhabited the oocyst sporulation in a dose-dependent manner. By day 5 after infection with the Eimeria parasite, the number of oocysts in mice feces was significantly reduced after RRE treatment (1.308 × 106 ± 1.36 × 105 oocysts/g feces) compared to the infected group (5.387 × 106 ± 4.29 × 105 oocysts/g feces). Moreover, the Eimeria infection reduced the number of goblet cells of mice jejuna and its specific gene, MUC2. The treatment with RRE increased the number of goblet cells/villus from 3.45 ± 0.17 to 6.04 ± 0.23, associated with upregulation for MUC2 from 0.26 to 2.39-fold. Also, the Eimeria experimental infection lowered the activity of the antioxidant enzyme represented by GPx (23.99 ± 3.68 mg/g tissue), while increasing the stress parameters of hydrogen peroxide (0.07 ± 0.01 mM/g) as well as the activity of MPO (66.30 ± 3.74 U/mg). The production of apoptotic markers including Caspase-3 (68.89 ± 2.67 U/g) and Bax (159.05 ± 6.50 pg/ml) was significantly elevated while decreasing the anti-apoptotic marker of BCL2 (0.42 ± 0.07 pg/ml). Our study proved that RRE significantly reduced oxidative stress, and apoptotic markers as well as the inflammatory activity of MPO. Also, antioxidant enzyme and anti-apoptotic activity in the jejunum of E. papillata-infected mice were enhanced after RRE treatment. Our study highlights the potential of RRE as a natural solution for coccidiosis management by modulating apoptosis in E. papillata host cells. However, further research is needed to fully understand the underlying mechanisms and enhance our understanding of its therapeutic efficacy.

Expression of IL-1β in transgenic Eimeria necatrix enhances the immunogenicity of parasites and promotes mucosal immunity against coccidiosis.

Anticoccidial vaccines comprising living oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima, and Eimeria acervulina are used to control coccidiosis. This study explored the potential of IL-1β to act as a molecular adjuvant for enhancing the immunogenicity of Eimeria necatrix and mucosal immunity. We engineered E. necatrix to express a functional chIL-1β (EnIL-1β) and immunized chickens with oocysts of the wild type (EnWT) and tranegenic (EnIL-1β) strains, respectively. The chickens were then challenged with EnWT oocysts to examine the immunogenicity-enhancing potential of chIL-1β. As expected, the oocyst output of EnIL-1β-immunized chickens was significantly reduced compared to those immunized using EnWT. No difference in body weight gain and lesion scores of EnIL-1β and EnWT groups was observed. The parasite load in the small intestine and caeca showed that the invasion and replication of EnIL-1β was not affected. However, the markers of immunogenicity and mucosal barrier, Claudin-1 and avian β-defensin-1, were elevated in EnIL-1β-infected chickens. Ectopic expression of chIL-1β in E. necatrix thus appears to improve its immunogenicity and mucosal immunity, without increasing pathogenicity. Our findings support chIL-1β as a candidate for development of effective live-oocyst-based anticoccidial vaccines.

Protective Efficacy Induced by the Common Eimeria Antigen Elongation Factor 2 against Challenge with Three Eimeria Species in Chickens.

Avian coccidiosis arises from co-infection involving multiple Eimeria species, which could give rise to substantial economic losses in the global poultry industry. As a result, multivalent anticoccidial vaccines containing common Eimeria antigens offer considerable promise for controlling co-infection in clinical practice. In our previous study, Elongation factor 2 (EF2) was deemed as an immunogenic common antigen across various Eimeria species. This current investigation aimed to further assess the immunogenicity and protective efficacy of EF2 in recombinant subunit vaccine format against three Eimeria species. The EF2 gene cloned from Eimeria maxima (E. maxima) cDNA was designated as EF2 of E. maxima (EmEF2). The immunogenicity of the recombinant protein EmEF2 (rEmEF2) was assessed through Western blot analysis. The evaluation of the vaccine-induced immune response encompassed the determination of T lymphocyte subset proportions, cytokine mRNA transcription levels, and specific IgY concentrations in rEmEF2-vaccinated chickens using flow cytometry, quantitative real-time PCR (qPCR), and indirect enzyme-linked immunosorbent assay (ELISA). Subsequently, the protective efficacy of rEmEF2 was evaluated through vaccination and challenge experiments. The findings demonstrated that rEmEF2 was effectively recognized by the His-tag monoclonal antibody and E. maxima chicken antiserum. Vaccination with rEmEF2 increased the proportions of CD4+ and CD8+ T lymphocytes, elevated IL-4 and IFN-γ mRNA transcription levels, and enhanced IgY antibody levels compared to the control groups. Moreover, compared to the control groups, vaccination with rEmEF2 led to decreased weight loss, reduced oocyst outputs, and alleviated enteric lesions. Furthermore, in the rEmEF2-immunized groups, challenges with E. maxima and E. acervulina resulted in anticoccidial index (ACI) scores of 166.35 and 185.08, showing moderate-to-excellent protective efficacy. Nevertheless, challenges with E. tenella and mixed Eimeria resulted in ACI scores of 144.01 and 127.94, showing low protective efficacy. In conclusion, EmEF2, a common antigen across Eimeria species, demonstrated the capacity to induce a significant cellular and humoral immune response, as well as partial protection against E. maxima, E. acervulina, and E. tenella. These results highlight EmEF2 as a promising candidate antigen for the development of multivalent vaccines targeting mixed infections by Eimeria species.

Protective efficacy of multiepitope vaccines constructed from common antigens of Eimeria species in chickens

Clinical avian coccidiosis is typically caused by coinfection with several Eimeria species. Recombinant protein and DNA vaccines have shown promise in controlling coccidiosis. On this basis, DNA vaccines that encode multiple epitopes from different Eimeria species may provide broad protection against coinfections. In this study, we designed a fusion gene fragment, 14EGT, that contained concentrated T-cell epitopes from four common antigens of Eimeria species (14-3-3, elongation factor 2, glyceraldehyde-3-phosphate dehydrogenase, and transhydrogenase). The multiepitope DNA vaccine pVAX1-14EGT and recombinant protein vaccine pET-32a-14EGT (r14EGT) were then created based on the 14EGT fragment. Subsequently, cellular and humoral immune responses were measured in vaccinated chickens. Vaccination-challenge trials were also conducted, where the birds were vaccinated with the 14EGT preparations and later exposed to single or multiple Eimeria species to evaluate the protective efficacy of the vaccines. According to the results, vaccination with 14EGT preparations effectively increased the proportions of CD4+ and CD8+ T cells and the levels of Th1 and Th2 hallmark cytokines. The levels of serum IgG antibodies were also significantly increased. Animal vaccination trials revealed alleviated enteric lesions, weight loss, and oocyst output compared to those of the control groups. The preparations were found to be moderately effective against single Eimeria species, with the anticoccidial index (ACI) ranging from 160 to 180. However, after challenge with multiple Eimeria species, the protection provided by the 14EGT preparations was not satisfactory, with ACI values of 142.18 and 146.41. Collectively, the results suggest that a multiepitope vaccine that encodes the T-cell epitopes of common antigens derived from Eimeria parasites could be a potential and effective strategy to control avian coccidiosis.

A rhoptry protein, localizing in the bulb region of rhoptries, could induce protective immunity against Eimeria tenella infection.

Rhoptry organelle proteins (ROPs) secreted by apicomplexan parasites play important roles during parasites invasion and survival in host cells, and are potential vaccine candidates against apicomplexan diseases. Eimeria tenella (E. tenella) is one of the most noteworthy apicomplexan species, which causes hemorrhagic pathologies. Although dozens of putative E. tenella ROP sequences are annotated, most ROP proteins are not well studied. In this study, an E. tenella ROP21 gene was identified and the recombinant EtROP21 protein (rEtROP21) was expressed in Escherichia coli. The developmental expression levels, localization, and protective efficacy against E. tenella infection in chickens were studied. An EtROP21 gene fragment with an open reading frame (ORF) of 981 bp was obtained from the Beijing strain of E. tenella. The rEtROP21 has a molecular weight of approximately 50 kDa and was recognized by rEtROP21-immunized mouse serum. Two specific protein bands, about 43 KDa and 95 KDa in size, were detected in the whole sporozoite proteins using the rEtROP21-immunized chicken serum. RT-qPCR analysis of the E. tenella ROP21 gene (EtROP21) revealed that its mRNA levels were higher in merozoites and sporozoites than in sporulated and unsporulated oocysts. Immunofluorescence and immunoelectron analyses showed that the EtROP21 protein predominantly localizes in the bulb region of rhoptries distributed at anterior, posterior, and perinuclear regions of E. tenella sporozoites. Immunization and challenge experiments revealed that immunizing chickens with rEtROP21 significantly increased their average body weight gain while decreasing mean lesion score and oocyst output (P <0.05). When compared with the challenged control group, the rEtROP21-immunized group was associated with a significantly higher relative weight gain (90.2%) and a greater reduction in oocyst output (67%) (P <0.05). The anticoccidial index of the rEtROP21-immunized group was 163.2. Chicken serum ELISA revealed that the levels of the specific anti- rEtROP21 antibody, IFN-γ, and IL-4 were significantly higher in the rEtROP21-immunized group than in the challenged control group (P <0.05). These results indicate that rEtROP21 can induce a high level of specific immune response and it is a potential candidate for the development of vaccines against E. tenella infection in chickens.