Tenella Infection Research Articles

Microbiota promotes recruitment and pro-inflammatory response of caecal macrophages during E. tenella infection

BackgroundEimeria genus belongs to the apicomplexan parasite phylum and is responsible for coccidiosis, an intestinal disease with a major economic impact on poultry production. Eimeria tenella is one of the most virulent species in chickens. In a previous study, we showed a negative impact of caecal microbiota on the physiopathology of this infection. However, the mechanism by which microbiota leads to the physiopathology remained undetermined. Macrophages play a key role in inflammatory processes and their interaction with the microbiota during E. tenella infection have never been investigated. We therefore examined the impact of microbiota on macrophages during E. tenella infection. Macrophages were monitored in caecal tissues by immunofluorescence staining with KUL01 antibody in non-infected and infected germ-free and conventional chickens. Caecal cells were isolated, stained, analyzed and sorted to examine their gene expression using high-throughput qPCR.ResultsWe demonstrated that microbiota was essential for caecal macrophage recruitment in E. tenella infection. Furthermore, microbiota promoted a pro-inflammatory transcriptomic profile of macrophages characterized by increased gene expression of NOS2, ACOD1, PTGS2, TNFα, IL1β, IL6, IL8L1, IL8L2 and CCL20 in infected chickens. Administration of caecal microbiota from conventional chickens to germ-free infected chickens partially restored macrophage recruitment and response.ConclusionsTaken together, these results suggest that the microbiota enhances the physiopathology of this infection through macrophage recruitment and activation. Consequently, strategies involving modulation of the gut microbiota may lead to attenuation of the macrophage-mediated inflammatory response, thereby limiting the negative clinical outcome of the disease.

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.

Dietary Oyster Mushroom (Pleurotus ostreatus) Waste Inhibits Experimentally Induced Eimeria tenella Challenge in Japanese Quails Model.

The aim of this study was to investigate the potential of dietary 3% oyster mushroom (Pleurotus ostreatus) waste in enhancing the anticoccidial effects in broilers challenged with Eimeria tenella infection. The experiment involved a total of 600 Japanese quails, raised from one to thirty-five days of age, which were divided into four treatment groups. These included a negative control group that received a basal diet (BD) without any anticoccidial or antibiotic supplementation in the non-challenged birds (negative control, NC); a positive control (PC) group consisting of NC birds challenged with E. tenella; a group that received the BD with an anticoccidial drug (standard); and a group that received the BD supplemented with 3% waste from oyster mushrooms (3% Pleurotus ostreatus). The results showed that the feed intake, body weight gain, and feed efficiency were significantly lower in the PC (p < 0.05). However, the growth traits were similar in the standard and 3% Pleurotus ostreatus-treated groups. Similarly, there was no difference (p < 0.05) in the mortality rate, oocyst count in the feces, and lesion score between the standard and 3% Pleurotus ostreatus groups. Based on intestinal histology evaluation, the villi height and width were significantly higher in the standard and 3% Pleurotus ostreatus-treated groups compared to those of the PC (p < 0.01). In conclusion, it was found that 3% Pleurotus ostreatus effectively mitigated the low growth rate of Japanese quails induced by coccidial infection.

Probiotic Enterococcus faecalis surface-delivering key domain of EtMIC3 proteins: immunoprotective efficacies against Eimeria tenella infection in chickens.

Enterococcus faecalis MDXEF-1 strain was used as host bacteria to deliver surface-displayed key domains BC1 and C4D of the Eimeria tenella microneme 3 (EtMIC3) protein, BC1 fusion to dendritic cell (DC) targeting peptides (DCpep) (DC-BC1) and DC-C4D, respectively. The expression of target proteins in MDXEF-1/BC1-CWA, MDXEF-1/DC-BC1-CWA, MDXEF-1/C4D-CWA, and MDXEF-1/DC-C4D-CWA was verified. The systemic immune responses induced by oral immunization with recombinant E. faecalis were recorded. The immunized chickens were challenged with 4.0 × 104 E. tenella-sporulated oocysts. The immune protections were evaluated by calculating average weight gain and oocyst reduction rate, observing gross and histopathological changes in the cecal tissues, and quantifying levels of inflammatory cytokines in the cecal tissues. The results showed that proteins BC1, C4D, DC-BC1, and DC-C4D were expressed on the surface of E. faecalis, respectively. Oral immunizations with recombinant E. faecalis, especially MDXEF-1/DC-BC1-CWA and MDXEF-1/DC-C4D-CWA, stimulated higher levels of antigen-specific humoral and intestinal mucosal immune responses, higher levels of Th1, Th2, and Th17-type cytokines, and higher proliferation of peripheral blood lymphocytes than phosphate-buffered saline (PBS) and vector control groups (P < 0.01). The groups immunized with four recombinant E. faecalis showed better protective effects against homologous infection than the vector control and infection control groups, and the MDXEF-1/DC-BC1-CWA group displayed the best effects. These results demonstrated that probiotic E. faecalis delivering DCpep fused with the key domain of the EtMIC3 protein could be a potential approach for the prevention of Eimeria infection. IMPORTANCE Avian coccidiosis caused by Eimeria brings huge economic losses to the poultry industry. Although live vaccines and anti-coccidial drugs were used for a long time, Eimeria infection in chicken farms all over the world commonly occurred. The exploration of novel, effective vaccines has become a research hotspot. Eimeria parasites have complex life cycles, and effective antigens are particularly critical to developing anti-coccidial vaccines. Microneme proteins (MICs), secreted from microneme organelles located at the parasite apex, are considered immunodominant antigens. Eimeria tenella microneme 3 (EtMIC3) contains four conserved repeats (MARc1, MARc2, MARc3, and MARc4) and three divergent repeats (MARa, MARb, and MARd), which play a vital role during the Eimeria invasion. Enterococcus faecalis is a native probiotic in animal intestines and can regulate intestinal flora. In this study, BC1 and C4D domains of EtMIC3, BC1 or C4D fusing to dendritic cells targeting peptides, were surface-displyed by E. faecalis, respectively. Oral immunizations were performed to investigate immune protective effects against Eimeria infection.

Evaluation of the Medicinal Potentials of Soursop (Annona muricata Safford) Leaf Extracts against Eimeria tenella Infection in Broiler Chickens

Aims: Poultry industry in Nigeria has recorded considerable expansion in recent times but not without poultry diseases as one of the major threats to boosting poultry production. Protozoa cause the majority of parasitic infections and cause high mortalities. Parasitic diseases such as coccidiosis are of particular importance because of their high incidence in poultry occasioned by the tropical environmental conditions under which the farmer operates. The resistance to common anticoccidial drugs has led to exploring other natural sources for prophylaxis and therapy. This research evaluated the therapeutic efficacies of Annona muricata at different dosages of 100, 125, 165mg/kg body weight using 120 broilers at 4 weeks of age. &#x0D; Place and Duration of Study: Animal Parasitology and Microbiology Research Unit, Department of Animal Production and Health, Federal University of Technology, Akure, Nigeria, between September and December, 2022.&#x0D; Methodology: 120 broilers of 4 weeks old were used for this study using completely randomized study design. The birds were infected with sporulated Eimeria tenella oocysts at week 4. Treatment with ethanolic extract of Annona muricata was administered orally at week 5 using different dosages of 100, 125, 165mg/kg body weight for five consecutive days. Treatment commenced after the isolation of Eimeria oocysts in the faeces and onset on coccidial clinical signs as parameters to confirm manifestation of the infection. Blood parameters, faecal oocysts count and biochemical assays were conducted.&#x0D; Results: Results revealed that therapeutic administration of Annona muricata leaf extracts at minimum dose 100mg/kg body weight had significant reduction (P&lt;0.05) on excreted Eimeria oocysts counts without any significant deviation from normal haematological and biochemical indices of the broilers.&#x0D; Conclusion: This study suggests the use of Annona muricata in the treatment of coccidiosis in broiler chickens. It is therefore recommended that its extracts be further explored for actual phytochemical constituents responsible for the displayed anticoccidial properties.

Transcriptomic, 16S ribosomal ribonucleic acid and network pharmacology analyses shed light on the anticoccidial mechanism of green tea polyphenols against Eimeria tenella infection in Wuliangshan black-boned chickens

BackgroundEimeria tenella is an obligate intracellular parasitic protozoan that invades the chicken cecum and causes coccidiosis, which induces acute lesions and weight loss. Elucidating the anticoccidial mechanism of action of green tea polyphenols could aid the development of anticoccidial drugs and resolve the problem of drug resistance in E. tenella.MethodsWe constructed a model of E. tenella infection in Wuliangshan black-boned chickens, an indigenous breed of Yunnan Province, China, to study the efficacy of green tea polyphenols against the infection. Alterations in gene expression and in the microbial flora in the cecum were analyzed by ribonucleic acid (RNA) sequencing and 16S ribosomal RNA (rRNA) sequencing. Quantitative real-time polymerase chain reaction was used to verify the host gene expression data obtained by RNA sequencing. Network pharmacology and molecular docking were used to clarify the interactions between the component green tea polyphenols and the targeted proteins; potential anticoccidial herbs were also analyzed.ResultsTreatment with the green tea polyphenols led to a reduction in the lesion score and weight loss of the chickens induced by E. tenella infection. The expression of matrix metalloproteinase 7 (MMP7), MMP1, nitric oxide synthase 2 and ephrin type-A receptor 2 was significantly altered in the E. tenella infection plus green tea polyphenol-treated group and in the E. tenella infection group compared with the control group; these genes were also predicted targets of tea polyphenols. Furthermore, the tea polyphenol (-)-epigallocatechin gallate acted on most of the targets, and the molecular docking analysis showed that it has good affinity with interferon induced with helicase C domain 1 protein. 16S ribosomal RNA sequencing showed that the green tea polyphenols had a regulatory effect on changes in the fecal microbiota induced by E. tenella infection. In total, 171 herbs were predicted to act on two or three targets in MMP7, MMP1, nitric oxide synthase 2 and ephrin type-A receptor 2.ConclusionsGreen tea polyphenols can directly or indirectly regulate host gene expression and alter the growth of microbiota. The results presented here shed light on the mechanism of action of green tea polyphenols against E. tenella infection in chickens, and have implications for the development of novel anticoccidial products.Graphical

Impacts of Eimeria coinfection on growth performance, intestinal health and immune responses of broiler chickens

Coccidiosis caused by Eimeria is one of the most severe chicken diseases and imposes huge economic losses to the poultry industry globally. Multi-Eimeria species coinfections are common with the most prevalent combination being mixtures of Eimeria acervulina and Eimeria tenella. Although detrimental impacts of either E. acervulina or E. tenella on chicken health are well recognized, no information is available regarding their coinfection effects so far. This study was designed to investigate the influence of coinfection with E. acervulina and E. tenella on broiler chickens. 144 one-day-old broiler chickens within each of trials (trial I or II) were divided into four groups, namely, control group (CG), E. acervulina infection group (EAIG), E. tenella infection group (ETIG) and dual (E. acervulina and E. tenella) infection group (DIG). Then, chickens were measured for weight loss, lesion scores, oocyst outputs, histological changes and expressions of pro-inflammatory (interleukin [IL]−6, IL-8 and IL-18), regulatory (IL-10 and IL-22) cytokines and Toll-like receptors (TLR; TLR2 and TLR4) as well as intestinal barrier (mucin 2 [MUC2] and fattey acid-bingding proteins 2 and 6 [FABP2 and FABP6])- and tight junction (TJ; zonula occluden-1 [ZO-1], occludin [OCLN], and claudins 1 and 5 [CLDN1 and CLDN5])-related proteins at 3, 5, 7, 10, 14 and 21 days post-infection, respectively. Our results consistently showed that although ETIG and DIG exhibited a higher level of weight loss and a more amount of oocyst excretion than EAIG, DIG had lighter lesions than EAIG in the early phase because of coinfection with E. tenella. A higher (P < 0.05) ratio of duodenal villous height to crypt depth was also observed in DIG than EAIG. Moreover, histological changes in the duodenum and cecum varied by single and dual Eimeria infections. Expressions of the intestinal barrier- and TJ-related genes of EAIG, ETIG and DIG were significantly (P < 0.05) upregulated but their levels exhibited differential changes among infected chickens. Similarly, the infected chickens showed significant (P < 0.05) inflammatory responses and higher (P < 0.05) expressions of TLRs in the intestines in comparison to CG. These results presented a comprehensive physiological, pathological and immunological characterization of E. acervulina and E. tenella coinfection in broiler chickens and also shed insights into pathogenesis of multi-coccidia coinfections.

The caecal microbiota promotes the acute inflammatory response and the loss of the intestinal barrier integrity during severe Eimeria tenella infection.

Coccidiosis, a disease caused by intestinal apicomplexan parasites Eimeria, is a threat to poultry production. Eimeria tenella is one of the most pathogenic species, frequently causing a high prevalence of opportunistic infections. The objective of this study is to investigate the role of the microbiota in the pathogenesis of severe Eimeria tenella infection. We have previously shown that microbiota can promote parasite development. To study the effect of the microbiota on the pathogenesis of this infection, we used an experimental condition (inoculum of 10 000 oocysts E. tenella INRAE) in which the parasite load is similar between germ-free and conventional broilers at 7 days post-infection (pi). Thirteen conventional and 24 germ-free chickens were infected. Among this latter group, 12 remained germ-free and 12 received a microbiota from conventional healthy chickens at 4 days pi. Caeca and spleens were collected at 7 days pi. Our results demonstrated caecal lesions and epithelium damage in conventional chickens at 7 days pi but not in germ-free infected chickens. Administration of conventional microbiota to germ-free chickens partially restored these deleterious effects. At day 7 pi, both infected conventional and germ-free chickens exhibited increased gene expression of inflammatory mediators, including IL15, IFNγ, TNFα and the anti-inflammatory mediator SOCS1, whereas the inflammatory mediators CXCLi2, CCL20, IL18, CSF1, NOS2, PTGS2, IL1β, IL6, the receptor CCR2, and the anti-inflammatory mediators TGFβ1 and IL10 were upregulated only in infected conventional chickens. Notably, the IL18, PTGS2 gene expression was significantly higher in the infected conventional group. Overall, the inflammatory response enhanced by the microbiota might be in part responsible for higher lesion scores. Epithelial tight junction protein gene expression analysis revealed a significant upregulation of CLDN1 with the infection and microbiota, indicating a potential loss of the intestinal barrier integrity. These observations imply that, during E. tenella infection, the caecal microbiota could trigger an acute inflammatory response, resulting in a loss of intestinal integrity. Increase in bacterial translocation can then lead to the likelihood of opportunistic infections. Hence, modulating the microbiota may offer a promising strategy for improving poultry gut health and limiting caecal coccidiosis.

Response to dietary methionine and organic zinc in broilers against coccidia under Eimeria tenella-challenged condition

The primary objective of this study is to explore whether organic zinc (OZn) and augmenting dietary methionine (Met) levels can enhance the anticoccidial effects in broilers when subjected to Eimeria tenella infection. Coccidiosis, induced by E. tenella. A total of 280 one-day-old Hubbard broilers in a coccidia-free environment were divided into seven groups. The first group served as the negative control and the second group E. tenella challenged. The third group was infected + amprolium treated. The fourth group was infected +50 mg/kg OZn. The fifth group was challenged and +0.56% Met. The sixth group was infected +OZn (50 mg/kg) + 0.56% Met. The seventh group was infected + OZn (25 mg/kg) + 0.28% Met. Growth performance was superior in combination of OZn (50 mg/kg) and 0.56% Met compared to positive control. Mortality, lesion score and oocyst count were significantly (P < 0.05) higher in positive control but significantly improved in OZn 50 mg/kg and 0.56% Met. Cecal morphological parameters were improved in OZn 50 mg/kg and 0.56% Met. We concluded that OZn 50 mg/kg and 0.56% Met improved growth performance of chickens under E. tenella challenge. From the results of the present study, it was concluded that OZn (50 mg/kg) in combination of methionine (0.56%) supplementation resulted in attenuation of negative effect of coccidial challenge and maintained the growth of broilers.

Natural Magnolol ameliorates coccidiosis infected with Eimeria tenella by affecting antioxidant, anti-inflammatory, and gut microbiota of chicks

Magnolol, a natural extract from magnolia officinalis, has received growing interest in its bioactive properties such as antioxidant, anti-inflammatory, and antibacterial activities. Nevertheless, there is little research on Magnolol in the treatment of parasitic infections currently. Eimeria tenella (E. tenella) infection causes damage to epithelial cells and cecal mucosa, resulting in increased intestinal permeability, which is pretty detrimental to the balance of the intestinal microenvironment. However, at present, in the treatment of chicken coccidiosis, the abuse of antibiotics is quite serious, which has brought losses and harms to the chicken farming industry that cannot be ignored. In this study, based on the excellent antioxidant and anti-inflammatory properties of Magnolol, we proved that it does have a desirable therapeutic potential on chicks infected with E. tenella. Actually, the results showed that the clinical symptoms of the chicks infected with E. tenella were relieved and their growth performance was restored by Magnolol treatment. Furthermore, Magnolol improved the antioxidant and anti-inflammatory properties of chicks. Meanwhile, the Magnolol reversed the imbalance of the intestinal microbiota of sick chicks, which recovered the diversity, promoted the potential beneficial bacteria, and inhabited the potential pathogenic bacteria. Overall, Magnolol may be an alternative to chemical drugs that are effective in treating E. tenella infections.

In-vivo anticoccidial efficacy of green synthesized iron-oxide nanoparticles using Ficus racemosa Linn leaf extract. (Moraceae) against Emeria tenella infection in broiler chicks

Coccidiosis is an acute gastrointestinal parasitic disease and causes approximately $2.80 to $3.27 per m2 loss in a broiler farm of a 33-day-old flock. In this study, iron oxide nanoparticles (IONPs) were green synthesized using the aqueous leaf extract of Ficus racemosa as a reducing and capping agent to reduce the emerging resistance in coccidia spores against conventional treatments and boost the immune level in broilers. These IONPs were evaluated for their impacts on the growth performance, biochemistry, blood profile, and histology in the coccidiodized broiler chicken with Emeria tenella under in vivo conditions. The characteristics and stability of particles were obtained using UV-Vis spectroscopy, Fourier transforms infrared (FTIR), X-Ray diffraction (XRD), energy dispersive X-ray absorption (EDX), scanning electron microscopy (SEM), zeta potential and zeta size. The results indicated that IONPs at the moderate dose of 15 mg/kg (p = 0.001) reduced the coccidial impacts by eliminating oocyst shedding per gram feces (up to 91%) and reducing clinical symptoms (lesions (LS = 0), bloody diarrhea (No), and mortality (0%) in chicken at day 10 of treatment as compared to the negative control group-B (infected & non-treated). A dose-dependent and time-dependent trend were observed during treatments (10, 15, and 20 mg/kg) of 1–3 weeks using IONPs against the coccidial impacts on the growth parameters (body weight gain, mean feed consumption, feed conversion ratio) and biochemistry (plasma glucose, total protein, uric acid, ALT, AST, and ALP) in chickens. Additionally, F. racemosa IONPs at a dose of 15 and 20 mg/kg significantly recovered the parasitized and highly damaged hepatocytes, liver tissues, and ceca tissues after 1–3 weeks of treatment in broiler chickens. Overall, the 15 mg/kg concentration of IONPs exhibited fast recovery and growth enhancement in coccidiodized broilers. Therefore, the 15 mg/kg dose of green synthesized IONPs using leaf extract of F. racemosa could be a potential and safe anticoccidial agent with targeted implications in the poultry industry.

Molecular characterization and protective efficacy of vacuolar protein sorting 29 from Eimeria tenella.

Vacuolar protein sorting 29 (VPS29) is a core component of the retromer-retriever complex and is essential for recycling numerous cell-surface cargoes from endosomes. However, there are no reports yet on VPS29 of Eimeria spp. Here, we cloned and prokaryotically expressed a partial sequence of Eimeria tenella VPS29 (EtVPS29) with RT-PCR and engineered strain of Escherichia coli respectively. The localization of the VPS29 protein in E. tenella sporozoites was investigated with immunofluorescence (IFA) and overexpression assays. And its protective efficacy against E. tenella infection was investigated in chickens with the animal protection test. An EtVPS29 gene fragment with an ORF reading frame of 549 bp was cloned. The band size of the expressed recombinant protein, rEtVPS29, was approximately 39 kDa and was recognized by the chicken anti-E. tenella positive serum. EtVPS29 protein was observed widely distributing in the cytoplasm of E. tenella sporozoites in the IFA and overexpression assays. rEtVPS29 significantly increased average body weight gain and decreased mean lesion score and oocyst output in chickens. The relative weight gain rate in the rEtVPS29-immunized group was 62.9%, which was significantly higher than that in the unimmunized and challenged group (P < 0.05). The percentage of reduced oocyst output in the rEtVPS29 immunized group was 32.2%. The anticoccidial index of the rEtVPS29-immunized group was 144.2. Serum ELISA also showed that rEtVPS29 immunization induced high levels of specific antibodies in chickens. These results suggest that rEtVPS29 can induce a specific immune response and is a potential candidate for the development of novel vaccines against E. tenella infections in chickens.

Comprehensive analysis of gut microbiome and host transcriptome in chickens after Eimeria tenella infection.

Coccidiosis is an intestinal parasitic disease caused by Eimeria protozoa, which endangers the health and growth of animals, and causes huge economic losses to the poultry industry worldwide every year. Studies have shown that poultry gut microbiota plays an important role in preventing the colonization of pathogens and maintaining the health of the host. Coccidia infection also affects host gene expression. However, the underlying potential relationship between gut microbiome and host transcriptome during E. tenella infection in chickens remain unclear. In this study, metagenomic and transcriptome sequencing were applied to identify microbiota and genes in cecal contents and cecal tissues of infected (JS) and control (JC) chickens on day 4.5 postinfection (pi), respectively. First, microbial sequencing results of cecal contents showed that the abundance of Lactobacillus, Roseburia sp. and Faecalibacterium sp decreased significantly after E. tenella infection (P < 0.05), while the abundance of Alistipes and Prevotella pectinovora increased significantly (P < 0.05). Second, transcriptome sequencing results showed that a total of 434 differentially expressed mRNAs were identified, including 196 up-regulated and 238 down-regulated genes. These differentially expressed genes related to inflammation and immunity, such as GAMA, FABP1, F2RL1 and RSAD2, may play an important role in the process of host resistance to coccidia infection. Functional studies showed that the enriched pathways of differentially expressed genes included the TGF-beta signaling pathway and the ErbB signaling pathways. Finally, the integrated analysis of gut microbiome and host transcriptome suggested that Prevotella pectinovora associated with FABP1, Butyricicoccus porcorum and Colidextribacter sp. associated with RSAD2 were involved in the immune response upon E. tenella infection. In conclusion, this study provides valuable information on the microbiota and key immune genes after chicken E. tenella infection, with the aim of providing reference for the impact of coccidia infection on cecal microbiome and host.

Pathogenicity and drug resistance of the Eimeria tenella isolate from Yiwu, Zhejiang province, eastern China

Chicken coccidiosis can cause severe enteritis with high mortality, which causes serious economic losses to the global breeding industry each year. The most virulent species is Eimeria tenella (E. tenella), but the infectivity of different E. tenella varies among geographic strains. At present, there are no reports related to the pathogenicity and drug resistance of E. tenella in Yiwu, Zhejiang province, China. A total of 600 fecal samples were collected from 10 farms in Zhejiang province, the overall oocyst prevalence was 54.2% (325/600). The prevalence was significantly higher (P < 0.01) in chickens under 40 d (97.5%) than that in chickens between 60 and 85-days-old (40.5%) and chickens over 90-days-old (24.5%). E. tenella stain was isolated from fecal samples of chickens in Yiwu and the pathogenicity of this isolate was determined, and then we recorded the survival rate, bloody stool score, lesion score, average weight gain. The results showed that all of the chickens infected with 5×105 sporulated oocysts of E. tenella died after the seventh day of infection, the bloody stool score and average lesion score of chickens from group 1 (5×105), group 2 (5×104), group 3 (5×103) and group 4 (5×102) decreased successively; the average weight gain (g) and relative weight gain (%) increased successively; the weight gain of the low-dose E. tenella infection groups (5×103 and 5×102) were higher than the other 2 groups (5×105 and 5×104) (P < 0.05). Finally, The E. tenella isolate was tested for sensitivity to 6 anticoccidial drugs (sulfachloropyrazine sodium, amproline, toltrazuril, clopidol, salinomycin, and nicarbazine) using 4 indexes including anticoccidial index(ACI), percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS), and relative oocyst production (ROP). The results showed that this isolate has developed severe resistance to drugs of salinomycin and nicarbazine, moderate resistance to amproline and clopidol, slight resistance to toltrazuril, while the E. tenella isolate performed more sensitive to sulfachloropyrazine sodium.

Development of a new immunodiagnostic tool for poultry coccidiosis using an antigen-capture sandwich assay based on monoclonal antibodies detecting an immunodominant antigen of Eimeria

This study was conducted to develop an antigen-capture ELISA that detects an immunodominant antigen of Eimeria, 3-1E which is present in all Eimeria species, using a set of 3-1E-specific mouse monoclonal antibodies (mAbs). Highly sensitive 3-1E-specific antigen-capture ELISA was established using compatible mAb pairs (#318 and #320) selected from 6 mAbs (#312, #317, #318, #319, #320, and #323) with high binding activity against recombinant 3-1E protein. These anti-3-1E mAbs specifically recognized E. tenella sporozoites and a higher level of 3-1E was detected in the lysate of sporozoites than in sporocysts. Immunofluorescence assay (IFA) using 2 mAbs (#318 and #320) showed specific staining around the membrane of E. tenella sporozoites. In order to measure the changes in the 3-1E level during in coccidiosis, serum, feces, jejunal, and cecal contents were individually collected daily for 7-days postinfection (dpi) with E. maxima and E. tenella. The new ELISA was sensitive and specific for 3-1E detection in all samples collected daily from E. maxima- and E. tenella-infected chickens for a week, and the detection sensitivity ranges were 2 to 5 ng/mL and 1 to 5 ng/mL in serum, 4 to 25 ng/mL and 4 to 30 ng/mL in feces, 1 to 3 ng/mL and 1 to 10 ng/mL in cecal contents, and 3 to 65 ng/mL and 4 to 22 ng/mL in jejunal contents. Following coccidiosis, the overall 3-1E levels started to increase from 4 dpi, and the highest production was shown on 5 dpi. Among the samples collected from Eimeria-infected chickens, the highest detection level was found in the jejunal contents of E. maxima-infected chickens. Furthermore, the level of IFN-γ in serum was significantly (P < 0.05) increased from 3 dpi and peaked on 5 dpi post E. maxima infection. Post E. tenella infection, the level of IFN-γ in serum gradually (P < 0.05) increased from 2 to 5 dpi and plateaued at 7 dpi. The level of TNF-α in serum was rapidly (P < 0.05) increased from 4 dpi and those levels were kept until 7 dpi post both Eimeria infections (E. maxima and E. tenella). More importantly, the daily changes in the 3-1E levels in different samples from E. maxima- and E. tenella-infected chickens were effectively monitored with this new antigen-capture ELISA. Therefore, this new immunoassay is a sensitive diagnostic tool to monitor coccidiosis in a large field population in the commercial poultry farms before clinical symptoms develop using serum, feces, and gut samples during the entire period of infection cycle starting from 1 d after infection.

Effect of zinc hydroxychloride supplementation combined with an anticoccidial drug on Eimeria tenella infection in broiler chickens.

Eimeria tenella is a causative agent of avian cecal coccidiosis resulting in bleeding, diarrhea, weight loss, high morbidity, and mortality in chickens. Zinc supplementation increases body weight gain, reduces mortality, and improves some immune response parameters of broilers infected with E. tenella. This study aimed to investigate the effects of zinc hydroxychloride (ZnOHCl) supplementation and ZnOHCl combined with an anticoccidial drug on E. tenella infection in broiler chickens. Forty one-day-old broilers were randomly divided into five groups, the study was replicated twice and had four chickens per replicate. Group 1 was an uninfected and unmedicated control group, and Group 2 was an infected but unmedicated control group. Group 3 was infected and treated with 120 mg/kg ZnOHCl, Group 4 was infected and medicated with 7 mg/kg toltrazuril (TOL), and Group 5 was infected and treated with 120 mg/kg ZnOHCl and 7 mg/kg TOL. Body weight gain, feed intake, and feed conversion ratio were monitored on days 15, 21, and 28. Oocyst shedding, hematological data, and lesion scores were analyzed on 7 days post-infection. The average weight gain, feed intake, and packed cell volume of chickens treated with ZnOHCl and TOL were significantly higher than those of the infected and unmedicated controls (p ≤ 0.05). Lesion scores, oocyst output, and lymphocytes of the chickens treated with ZnOHCl and TOL were significantly lower than those of the infected and unmedicated controls (p ≤ 0.05). This study demonstrated that zinc supplementation alone reduced only oocyst output. However, growth performance, lesion scores, and oocyst output were affected by the combination of ZnOHCl and TOL supplementation. This suggests that ZnOHCl supplementation combined with an anticoccidial drug can improve growth performance and lessen the severity of E. tenella infection.

Pathogenic mechanism of Eimeria tenella autophagy activation of chicken embryo cecal epithelial cells induced by Eimeria tenella

Eimeria tenella mainly invades and develops into cecal epithelial cells of chickens, resulting in cecal epithelial cell damage. Infectious intracellular pathogens possibly act by influencing the autophagy process after invading cells. The interaction between E. tenella and the autophagy of host cells was explored by infecting E. tenella with chick embryo cecal epithelial cells. Transmission electron microscopy, laser confocal microscopy, and Western blot analysis were used to demonstrate that E. tenella infection could induce autophagy in host cells. Results showed that infection with E. tenella induced the formation of autophagosomes in cells. The expression of ATG 5, Beclin-1, and LC3B-II proteins were significantly (P < 0.01) increased after E. tenella infected host cells. Expression of p62 protein levels were significantly (P < 0.01) decreased in host cells infected with E. tenella. Chloroquine (CQ) significantly (P < 0.01) increased the expression levels of LC3B-II and P62 in E. tenella-infected host cells. Rapamycin (RAPA) induced autophagy in host cells, thus reducing the intracellular infection of E. tenella. By contrast, the infection rate of E. tenella increased in cells treated with 3-Methyladenine (3-MA). Hence, E. tenella sporozoite infection could induce autophagy activation in chick embryo cecal epithelial cells, and enhanced autophagy could reduce the infection rate of E. tenella.

Phenotypic characterization of Eimeria tenella-specific chicken T-cells responding to in vitro parasite antigen re-stimulation.

Introduction. Coccidiosis, caused by protozoan parasites of genus Eimeria, is a disease with large impact on poultry production worldwide. It is well known that Eimeria immunity is dependent on Th1-type responses.Gap Statement. In vitro assessment of Eimeria-specific T-cell activity would therefore be a valuable research tool but has so far proven difficult to establish.Aim. The present study aimed to evaluate in vitro induced blast transformation and CD25 expression in defined chicken T-cell populations as a measure of Eimeria immunity.Methodology. Three E. tenella infection experiments were performed and PBMC and/or spleen cells were collected between 6 and 16 days after infection of chickens. Cells were stimulated in vitro with E. tenella antigens and T-cell activation was assessed by immunofluorescence labelling and flow cytometry.Results. The results consistently showed statistically significant E. tenella specific activation of TCRα/β+T cells within a 'window' from 8 to 14 days after infection for both spleen cells and PBMC. Responding T-cells were identified as CD4+CD8-, CD4+CD8αα+ and CD4-CD8αβ+ where the CD4+CD8αα+ cells generally showed the highest responses. All three of these TCRα/βT-cell subsets showed significant E. tenella induced blast transformation and/or CD25 expression albeit not always in concert on the same days after infection indicating complex kinetics of T-cell responses. In general, responses were higher for spleen cells compared to PBMC for all responding T-cell populations.Conclusions. This methodology shows promise to study Eimeria-specific T-cells, e.g. to evaluate vaccine responses. Results indicated that a Th1-type response was induced and suggested a role for CD4+CD8αα+ cells in Eimeria immunity.

Serum biochemistry indices, leukogram, carcass variables and intestinal measurements of Eimeria tenella-infected or non-infected broilers treated with dietary Cinnamomum verum bark

ABSTRACT We compared the outcomes of various concentrations of Cinnamomum verum bark powder (CNP), a natural product, to a conventional anticoccidial drug in induced Eimeria tenella infection in broilers aged 21 days. On day 21, 250 birds (Ross 308) were randomly assigned to one of 10 treatments, five of which had Eimeria tenella infection and five of which did not. Results 14 days post-infection (dpi) revealed that the treatments had a significant effect on body weight gain (BWG) and production efficiency factor (PEF). Although the 2 g CNP and Salinomycin (Sacox) treatments performed equally well, the 2 g CNP had the highest BWG and PEF under challenging conditions. Although the PEF improved satisfactorily at 14 dpi, the loss of BWG at 7 dpi did not compensate at 14 dpi. Challenged birds had longer and heavier small intestines, atrophiedceca, lower creatinine levels and higher eosinophils % than unchallenged birds. The elevation lesion score and oocyst secretion of challenged birds decreased with increasing cinnamon dosage. In conclusion, 6 g CNP as a natural product could be as effective as Sacox against occidiosis, with 2 g CNP performing best at 14 dpi. However, more research is required to fully understand its anticoccidial mechanisms. Highlights Cinnamomum verum bark may benefit broiler health by reducing Eimeria tenella oocysts. Under coccidial challenge, Cinnamomum verum bark at 2 g/kg diet may improve broiler body weight gain and production efficiency at 34 days of age (14 days post-infection). The lesion score and oocyst secretion of challenged birds decreased less as cinnamon dosage was increased.

Construction of a Lactobacillus plantarum-based claudin-3 targeting delivery system for the development of vaccines against Eimeria tenella.

Avian coccidiosis causes huge economic losses to the poultry industry worldwide and currently lacks effective live vector vaccines. Achieving efficient antigen delivery to mucosa-associated lymphoid tissue (MALT) is critical for improving the effectiveness of vaccines. Here, chicken claudin-3 (CLDN3), a tight junction protein expressed in MALT, was identified as a target, and the C-terminal region of Clostridium perfringens enterotoxin (C-CPE) was proven to bind to chicken CLDN3. Then, a CLDN3-targeting Lactobacillus plantarum NC8-expressing C-CPE surface display system (NC8/GFP-C-CPE) was constructed to successfully express the heterologous protein on the surface of L. plantarum. The colonization level of NC8/GFP-C-CPE was significantly increased compared to the non-targeting strain and could persist in the intestine for at least 72h. An oral vaccine strain expressing five EGF domains of Eimeria tenella microneme protein 8 (EtMIC8-EGF) (NC8/EtMIC8-EGF-C-CPE) was constructed to evaluate the protective efficacy against E. tenella infection. The results revealed that CLDN3-targeting L. plantarum induced stronger mucosal immunity in gut-associated lymphoid tissues (GALT) as well as humoral responses and conferred better protection in terms of parasite replication and pathology than the non-targeting strain. Overall, we successfully constructed a CLDN3-targeting L. plantarum NC8 surface display system characterized by MALT-targeting, which is an efficient antigen delivery system to confer enhanced protective efficacy in chickens against E. tenella infection.