Em14-3-3 delivered by PLGA and chitosan nanoparticles conferred improved protection in chicken against Eimeria maxima.

Eimeria maxima (E. maxima) are an intracellular apicomplexan protozoan that causes intestinal coccidiosis in chickens. The purpose of this research was to develop a novel delivery approach for recombinant E. maxima (rEm) 14-3-3 antigen to elicit enhanced immunogenic protection using poly (D, L-lactide-co-glycolide) (PLGA) and chitosan (CS) nanoparticles (NPs) against E. maxima challenge. The morphologies of prepared antigen-loaded NPs (PLGA/CS-rEm14-3-3 NPs) were visualized by a scanning electron microscope. The rEm14-3-3 and PLGA/CS-rEm14-3-3 NPs-immunized chicken-induced changes of serum cytokines, IgY-antibody level, and T-lymphocyte subsets and protective efficacies against E. maxima challenge were evaluated. The results revealed that encapsulated rEm14-3-3 in PLGA and CS NPs presented spherical morphology with a smooth surface. The chickens immunized with only rEm14-3-3 and PLGA/CS-rEm14-3-3 NPs elicited a significant (p<0.05) higher level of IFN-γ cytokine, stimulated the proportions of CD4+/CD3+, CD8+/CD3+ T-cells, and provoked sera IgY-antibody immune response compared to control groups (PBS, pET-32a, PLGA, and CS). Whereas, PLGA-rEm14-3-3 NP-immunized chicken provoked a higher level of IFN- γ production and IgY-antibody response rather than CS-rEm14-3-3 and bare antigen, relatively. The animal experiment results ratified that PLGA-rEm14-3-3 NP-immunized chicken significantly alleviated the relative body weight gain (%), decreased lesion score, and enhanced oocyst decrease ratio compared to CS-rEm14-3-3 NPs and only rEm14-3-3. The anti-coccidial index of the chicken vaccinated with the PLGA-rEm14-3-3 NPs was (180.1) higher than that of the Cs-rEm14-3-3 NPs (167.4) and bare antigen (165.9). Collectively, our statistics approved that PLGA NPs might be an efficient antigen carrier system (Em14-3-3) to act as a nanosubunit vaccine that can improve protective efficacies in chicken against E. maxima challenge.

Rhomboid protein 2 of Eimeria maxima provided partial protection against infection by homologous species.

Rhomboid-like proteases (ROMs) are considered as new candidate antigens for developing new-generation vaccines due to their important role involved in the invasion of apicomplexan protozoa. In prior works, we obtained a ROM2 sequence of Eimeria maxima (EmROM2). This study was conducted to evaluate the immunogenicity and protective efficacy of EmROM2 recombinant protein (rEmROM2) and EmROM2 DNA (pVAX1-EmROM2) against infection by Eimeria maxima (E. maxima). Firstly, Western blot assay was conducted to analyze the immunogenicity of rEmROM2. The result showed that rEmROM2 was recognized by chicken anti-E. maxima serum. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay revealed apparent transcription and expression of EmROM2 at the injection site. qRT-PCR (quantitative real-time PCR), flow cytometry and indirect ELISA indicated that vaccination with rEmROM2 or EmROM2 DNA significantly upregulated the transcription level of cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-17, TGF-ß and TNF SF15), the proportion of CD8+ and CD4+ T lymphocytes and serum IgG antibody response. Ultimately, a vaccination-challenge trial was performed to evaluate the protective efficacy of rEmROM2 and pVAX1-EmROM2 against E. maxima. The result revealed that vaccination with rEmROM2 or pVAX1-EmROM2 significantly alleviated enteric lesions, weight loss, and reduced oocyst output caused by challenge infection of E. maxima, and provided anticoccidial index (ACI) of more than 160, indicating partial protection against E. maxima. In summary, vaccination with rEmROM2 or pVAX1-EmROM2 activated notable humoral and cell-mediated immunity and provided partial protection against E. maxima. These results demonstrated that EmROM2 protein and DNA are promising vaccine candidates against E. maxima infection.

Impacts of increasing challenge with Eimeria maxima on the growth performance and gene expression of biomarkers associated with intestinal integrity and nutrient transporters.

This study was conducted to investigate the impacts of graded severity of Eimeria maxima infection on the growth performance and intestine health of broiler chickens. Four different levels of E. maxima-challenged treatments were used, including a non-challenged control group, a low challenge (12 500 oocysts), a medium challenge (25 000 oocysts), and a high challenge dose (50 000 oocysts). There were eight replicate cages per treatment, with 12 birds in each cage, and chickens in the challenged groups orally received sporulated oocysts on day 14. Gastrointestinal permeability was measured by fluorescein isothiocyanate dextran at 5 days post-infection (dpi), whereas intestinal morphology and gene expression of nutrient transporters and tight junction proteins were determined at 6 dpi. The results demonstrate a linear reduction in growth performance, jejunal villus height, and jejunal integrity with graded challenge doses of E. maxima (P < 0.01). Moreover, linear regulation of nutrient transporters and tight junction proteins was a consequence of increasing Eimeria infection levels (P < 0.01). The linear increase of Claudin 1, cationic amino acid transporter, glucose transporter 1, and L-type amino acid transporter genes was associated with increased severity of coccidiosis (P < 0.01). Furthermore, expression of nutrient transporters located at the brush border membrane were down-regulated (P < 0.01) with increasing E. maxima inoculation dose. In conclusion, growth performance and key intestinal integrity biomarkers in broiler chickens were adversely influenced in a dose-dependent manner by E. maxima infection.

Efficacy of recombinant N- and C-terminal derivative of EmIMP1 against E. maxima infection in chickens.

1. Immune mapped protein-1 (IMP1) of E. maxima has been identified as a vaccine antigen candidate for E. maxima infection. 2. In the current study, the N- and C-terminal derivative of EmIMP1 were expressed in E. coli and administered to chickens. The antibody response, cell-mediated immune (CMI) response and the protective efficacy of the protein vaccines against E. maxima challenge were evaluated. 3. The results showed that C-terminal derivative of EmIMP1 vaccination could increase weight gain, reduce enteric lesions, and decrease faecal oocysts shedding. Moreover, the C-terminal derivative of EmIMP1 caused reasonable improvement in serum antibodies and the numbers of IFN-γ producing peripheral blood mononuclear cells (PBMC), as compared to the control group. 4. This study demonstrated that the C-terminal derivative of EmIMP1 could be used as a potent immunogenic candidate in the development of subunit vaccines against E. maxima infection.

Dietary vitamin D improves performance and bone mineralisation, but increases parasite replication and compromises gut health in Eimeria-infected broilers.

Coccidial infections reduce fat-soluble vitamin status and bone mineralisation in broiler chickens. We hypothesised that broilers infected with Eimeria maxima would benefit from increased dietary supplementation with vitamin D (vitD) or with 25-hydroxycholecalciferol (25(OH)D3 or 25D3). Broilers were assigned to diets with low (L) or commercial (M) vitD levels (25 v. 100 µg/kg) supplemented as cholecalciferol (D3) or 25D3. At day 11 of age, birds were inoculated with water or 7000 E. maxima oocysts. Pen performance was calculated over the early (days 1-6), acute (days 7-10) and recovery periods (days 11-14) post-infection (pi). At the end of each period, six birds per treatment were dissected to assess long bone mineralisation, plasma levels of 25D3, Ca and P, and intestinal histomorphometry. Parasite replication and transcription of cytokines IL-10 and interferon-γ (IFN-γ) were assessed at day 6 pi using quantitative PCR. Performance, bone mineralisation and plasma 25D3 levels were significantly reduced during infection (P < 0·05). M diets or diets with 25D3 raised plasma 25D3, improved performance and mineralisation (P < 0·05). Offering L diets compromised feed efficiency pi, reduced femur breaking strength and plasma P levels at day 10 pi in infected birds (P < 0·05). Contrastingly, offering M diets or diets with 25D3 resulted in higher parasite loads (P < 0·001) and reduced jejunal villi length at day 10 pi (P < 0·01), with no effect on IL-10 or IFN-γ transcription. Diets with M levels or 25D3 improved performance and mineralisation, irrespective of infection, while M levels further improved feed efficiency and mineralisation in the presence of coccidiosis.

Comparative transcriptome analysis of Eimeria maxima (Apicomplexa: Eimeriidae) suggests DNA replication activities correlating with its fecundity.

BACKGROUND: Chicken coccidiosis, caused by the infection of Eimeria species, leads to important economic losses to the poultry industry. Vaccination with attenuated live parasites seems to be the best way to control this disease. Attenuated eimerian parasites with shortened prepatent times show great changes in intracellular development compared to their parent strains but the mechanisms involved in these biological differences are still unclear. RESULTS: In this study, we obtained a precocious line of E. maxima by sequential selection of 22 generations of early shed oocysts in chickens and performed a comparative transcriptome analysis of three different developmental stages of the precocious line and its parent strain using Illumina high-throughput sequencing. Our E. maxima precocious line showed decreased pathogenicity, reduced fecundity and a greatly shorted prepatent time of only 98 h. We found that typical gene changes in the stage development from unsporulated to sporulated oocyst and from sporulated oocyst to merozoite were marked by upregulated organelle genes and protein translation related genes, respectively. Additionally, major differences between the precocious line and its parent strain were detected in the merozoite stage, characterized by downregulated genes involved in protein cleavage and DNA replication activities. CONCLUSIONS: Our study generated and characterized an E. maxima precocious line, illustrating gene expression landscapes during parasite development by transcriptome analysis. We also show that the suppressed DNA replication progress in the merozoite stage in the precocious line may result in its reduced fecundity. These results provide the basis for a better understanding of the mechanism of precocity in Eimeria species, which can be useful in studies in early gametocytogenesis in apicomplexan parasites.

Re-evaluation of the life cycle of Eimeria maxima Tyzzer, 1929 in chickens (Gallus domesticus).

A time-course study was conducted to resolve discrepancies in the literature and better define aspects of the Eimeria maxima life cycle such, as sites of development and both morphology and number of asexual stages. Broiler chickens were inoculated orally with five million E. maxima oocysts (APU1), and were necropsied at regular intervals from 12 to 120 h p.i. Small intestine tissue sections and smears were examined for developmental stages. The jejunum contained the highest numbers of developmental stages. At 12 h p.i., sporozoites were observed inside a parasitophorous vacuole (PV) in the epithelial villi and the lamina propria. By 24 h, sporozoites enclosed by a PV were observed in enterocytes of the glands of Lieberkühn. At 48 h p.i., sporozoites, elongated immature and mature schizonts, were all seen in the glands with merozoites budding off from a residual body. By 60 h, second-generation, sausage-shaped schizonts containing up to 12 merozoites were observed around a residual body in the villar tip of invaded enterocytes. At 72 and 96 h, profuse schizogony associated with third- and fourth-generation schizonts was observed throughout the villus. At 120 h, another generation (fifth) of schizonts were seen in villar tips as well as in subepithelium where gamonts and oocysts were also present; a few gamonts were in epithelium. Our finding of maximum parasitization of E. maxima in jejunum is important because this region is critical for nutrient absorption and weight gain.

Expression of digestive enzymes and nutrient transporters in Eimeria-challenged broilers.

Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. The site of invasion and lesions in the intestine is species-specific, for example E. acervulina affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain. The growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to compare the expression of digestive enzymes, nutrient transporters and an antimicrobial peptide in broilers challenged with either E. acervulina, E. maxima or E. tenella. The genes examined included digestive enzymes (APN and SI), peptide and amino acid transporters (PepT1, ASCT1, b(0,+)AT/rBAT, B(0)AT, CAT1, CAT2, EAAT3, LAT1, y(+)LAT1 and y(+)LAT2), sugar transporters (GLUT1, GLUT2, GLUT5 and SGLT1), zinc transporter (ZnT1) and an antimicrobial peptide (LEAP2). Duodenum, jejunum, ileum and ceca were collected 7 days post challenge. E. acervulina challenge resulted in downregulation of various nutrient transporters or LEAP2 in the duodenum and ceca, but not the jejunum or ileum. E. maxima challenge produced both downregulation and upregulation of nutrient transporters and LEAP2 in all three segments of the small intestine and ceca. E. tenella challenge resulted in the downregulation and upregulation of nutrient transporters and LEAP2 in the jejunum, ileum and ceca, but not the duodenum. At the respective target tissue, E. acervulina, E. maxima and E. tenella infection caused common downregulation of APN, b(0,+)AT, rBAT, EAAT3, SI, GLUT2, GLUT5, ZnT1 and LEAP2. The downregulation of nutrient transporters would result in a decrease in the efficiency of protein and polysaccharide digestion and uptake, which may partially explain the weight loss. The downregulation of nutrient transporters may also be a cellular response to reduced expression of the host defense protein LEAP2, which would diminish intracellular pools of nutrients and inhibit pathogen replication.

RNA-seq Profiles of Immune Related Genes in the Spleen of Necrotic Enteritis-afflicted Chicken Lines.

The study aimed to compare the necrotic enteritis (NE)-induced transcriptome differences between the spleens of Marek's disease resistant chicken line 6.3 and susceptible line 7.2 co-infected with Eimeria maxima/Clostridium perfringens using RNA-Seq. Total RNA from the spleens of two chicken lines were used to make libraries, generating 42,736,296 and 42,617,720 usable reads, which were assembled into groups of 29,897 and 29,833 mRNA genes, respectively. The transcriptome changes were investigated using the differentially expressed genes (DEGs) package, which indicated 3,255, 2,468 and 2,234 DEGs of line 6.3, line 7.2, and comparison between two lines, respectively (fold change ≥2, p<0.01). The transcription levels of 14 genes identified were further examined using qRT-PCR. The results of qRT-PCR were consistent with the RNA-seq data. All of the DEGs were analysed using gene ontology terms, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the DEGs in each term were found to be more highly expressed in line 6.3 than in line 7.2. RNA-seq analysis indicated 139 immune related genes, 44 CD molecular genes and 150 cytokines genes which were differentially expressed among chicken lines 6.3 and 7.2 (fold change ≥2, p<0.01). Novel mRNA analysis indicated 15,518 novel genes, for which the expression was shown to be higher in line 6.3 than in line 7.2 including some immune-related targets. These findings will help to understand host-pathogen interaction in the spleen and elucidate the mechanism of host genetic control of NE, and provide basis for future studies that can lead to the development of marker-based selection of highly disease-resistant chickens.

CDR3 analysis of TCR Vß repertoire of CD8⁺ T cells from chickens infected with Eimeria maxima.

CD8(+) T cells play a major role in the immune protection of host against the reinfection of Eimeria maxima, the most immunogenic species of eimerian parasites in chickens. To explore the dominant complementarity-determining regions 3 (CDR3) of CD8(+) T cell populations induced by the infection of this parasite, sequence analysis was performed in this study for CDR3 of CD8(+) T cells from E. maxima infected chickens. After 5 days post the third or forth infection, intraepithelial lymphocytes were isolated from the jejunum of bird. CD3(+)CD8(+) T cells were sorted and subjected to total RNA isolation and cDNA preparation. PCR amplification and cloning of the loci between Vß1 and Cß was conducted for the subsequent sequencing of CDR3 of T cell receptor (TCR). After the forth infection, 2 birds exhibited two same frequent TCR CDR3 sequences, i.e., AKQDWGTGGYSNMI and AGRVLNIQY; while the third bird showed two different frequent TCR CDR3 sequences, AKQGARGHTPLN and AKQDIEVRGPNTPLN. No frequent CDR3 sequence was detected from uninfected birds, though AGRVLNIQY was also found in two uninfected birds. Our result preliminarily demonstrates that frequent CDR3 sequences may exist in E. maxima immunized chickens, encouraging the mining of the immunodominant CD8(+) T cells against E. maxima infection.

Differential regulation of microRNA transcriptome in chicken lines resistant and susceptible to necrotic enteritis disease.

Necrotic enteritis (NE) is a re-emerging disease as a result of increased restriction on the use of antibiotics in poultry. However, the molecular mechanisms underlying the pathogenesis of NE are unclear. Small RNA transcriptome analysis was performed using spleen and intestinal intraepithelial lymphocytes (IEL) from 2 inbred chicken lines selected for resistance or susceptibility to Marek's disease (MD) in an experimentally induced model of avian NE to investigate whether microRNA (miRNA) control the expression of genes associated with host response to pathogen challenge. Unique miRNA represented only 0.02 to 0.04% of the total number of sequences obtained, of which 544 were unambiguously identified. Hierarchical clustering revealed that most of miRNA in IEL were highly expressed in the MD-susceptible line 7.2 compared with MD-resistant line 6.3. Reduced CXCL14 gene expression was correlated with differential expression of several unique miRNA in MD-resistant chickens, whereas TGFßR2 gene expression was correlated with altered gga-miR-216 miRNA levels in MD-susceptible animals. In conclusion, miRNA profiling and deep sequencing of small RNA in experimental models of infectious diseases may be useful for further understanding of host-pathogen interactions, and for providing insights into genetic markers of disease resistance.

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.

Early Salmonella Typhimurium inoculation may obscure anti-interleukin-10 protective effects on broiler performance during coccidiosis and necrotic enteritis challenge.

Anti-interleukin (IL)-10 may preserve broiler performance during coccidiosis by diminishing Eimeria spp. host-evasion but has not been evaluated during secondary Clostridium perfringens challenge (necrotic enteritis). Early Salmonella Typhimurium inoculation is implemented in some models to improve repeatability-a potential confounder due to Salmonella using similar IL-10 host evasion pathways. The objective was to evaluate performance and disease outcomes in broilers fed anti-IL-10 during necrotic enteritis challenge ± S. Typhimurium. Three 42 d replicate studies in wire-floor cages (32 cages/replicate) were conducted with Ross 308 chicks assigned to diets ± 0.03% anti-IL-10 for 25 d before moving to floor pens for the study remainder. In replicates 1 and 2, 640 chicks were placed at hatch (20/cage) and inoculated with sterile saline ± 1 × 108 colony forming units (CFU) S. Typhimurium. Replicate 3 placed 480 chicks (15/cage) at hatch. On d 14, S. Typhimurium-inoculated chicks (replicates 1 and 2) or those designated for challenge (replicate 3) were inoculated with 15,000 sporulated Eimeria maxima M6 oocysts. On d 18 and 19, half the E. maxima-challenged chicks were gavaged with 1 × 108 CFU C. perfringens. Body weight (BW) and feed intake were measured throughout, while 6 chicks/ treatment were scored for jejunal lesions at 7 and 3 d postinoculation (pi) with E. maxima and C. perfringens, respectively. Oocyst shedding was measured at 8 and 4 dpi with E. maxima and C. perfringens, respectively. Performance and oocyst shedding were analyzed with diet and challenge fixed effects (SAS 9.4), whereas lesion scores and mortalities were analyzed by ordinal logistic regression (R 4.2.2; P ≤ 0.05). In replicate 3, no wk 3 feed conversion ratio (FCR) differences were observed between chicks fed anti-IL-10 challenged with E. maxima ± C. perfringens, whereas control-fed chicks had a 50 point less efficient FCR during E. maxima + C. perfringens challenge vs. E. maxima only (P = 0.04). Outcomes suggest anti-IL-10 may preserve bird feed efficiency during necrotic enteritis challenge in models without S. Typhimurium.

Sequencing approaches to identify distal jejunum microbial community composition and function in broiler chickens fed anti-interleukin-10 during coccidiosis and necrotic enteritis challenge.

Strategies to counteract interleukin (IL)-10-mediated immune evasion by Eimeria spp. during coccidiosis- like anti-IL-10 antibodies- may protect broiler chicken health and reduce incidence of secondary necrotic enteritis (Clostridium perfringens) via undetermined mechanisms. Objectives were to use sequencing techniques to evaluate jejunal microbial community composition and function in anti-IL-10-fed broilers during coccidiosis and necrotic enteritis. On d0, Ross 308 chicks were placed in 32 cages (15 chicks/ cage) for a 25-d study and randomly assigned to diets ± 0.03% anti-IL-10. Six chicks/ diet were euthanized for distal jejunum content and tissue collection on d 14 (baseline) before inoculating the remainder with saline or 15,000 E. maxima oocysts (M6 strain). Half the chicks challenged with E. maxima were challenged with C. perfringens (1×108 colony forming units) on d 18 and 19. Follow-up samples (6 chicks/treatment) were collected at 7 and 11 d postinoculation (pi) for the E. maxima-only group, or 3 and 7 dpi for the E. maxima + C. perfringens group with 3/7 dpi being designated as peak and 7/11dpi as postpeak challenge. DNA was extracted from digesta for microbiota composition analysis (16S rRNA gene sequencing) while RNA was extracted from tissue to evaluate the metatranscriptome (RNA sequencing). Alpha diversity and genus relative abundances were analyzed using the diet or challenge main effects with associated interactions (SAS 9.4; P ≤ 0.05). No baseline microbial changes were associated with dietary anti-IL-10. At peak challenge, a diet main effect reduced observed species 36.7% in chicks fed anti-IL-10 vs. control; however, the challenge effect reduced observed species and Shannon diversity 51.2-58.3% and 33.0 to 35.5%, respectively, in chicks challenged with E. maxima ± C. perfringens compared to their unchallenged counterparts (P ≤ 0.05). Low sequencing depth limited metatranscriptomic analysis of jejunal microbial function via RNA sequencing. This study demonstrates that challenge impacted the broiler distal jejunum microbiota more than anti-IL-10 while future research to characterize the microbial metatranscriptome may benefit from investigating other intestinal compartments.

Comprehensive analysis of differentially expressed mRNA profiles in chicken jejunum and cecum following Eimeria maxima infection.

Coccidiosis, a protozoan disease that substantially impacts poultry production, is characterized by an intracellular parasite. The study utilized 48 one-day-old Horro chickens, randomly divided into the infected (I) and control (C) groups. The challenge group of chickens were administered Eimeria maxima oocysts via oral gavage at 21-days-old, and each chicken received 2 mL containing 7×104 sporulated oocysts. The total RNAs of chicken jejunum and cecum tissues were isolated from three samples, each from I and C groups. Our study aimed to understand the host immune-parasite interactions and compare immune response mRNA profiles in chicken jejunum and cecum tissues at 4 and 7 days postinfection with Eimeria maxima. The results showed that 823 up- and 737 down-regulated differentially expressed mRNAs (DEmRNAs) in jejunum at 4 d infection and control (J4I vs. J4C), and 710 up- and 368 down-regulated DEmRNAs in jejunum at 7 days infection and control (J7I vs. J7C) were identified. In addition, DEmRNAs in cecum tissue, 1424 up- and 1930 down-regulated genes in cecum at 4 days infection and control (C4I vs. C4C), and 77 up- and 191 down-regulated genes in cecum at 7 days infection and control (C7I vs. C7C) were detected. The crucial DEmRNAs, including SLC7A5, IL1R2, GLDC, ITGB6, ADAMTS4, IL1RAP, TNFRSF11B, IMPG2, WNT9A, and FOXF1, played pivotal roles in the immune response during Eimeria maxima infection of chicken jejunum. In addition, the potential detection of FSTL3, RBP7, CCL20, DPP4, PRKG2, TFPI2, and CDKN1A in the cecum during the host immune response against Eimeria maxima infection is particularly noteworthy. Furthermore, our functional enrichment analysis revealed the primary involvement of DEmRNAs in small molecule metabolic process, immune response function, inflammatory response, and toll-like receptor 10 signaling pathway in the jejunum at 4 and 7 days postinfection. Similarly, in the cecum, DEmRNAs at 4 and 7 days postinfection were enriched in processes related to oxidative stress response and immune responses. Our findings provide new insights and contribute significantly to the field of poultry production and parasitology.

Coccidiosis and necrotic enteritis model may have a greater impact than dietary anti-interleukin-10 on broiler chicken systemic immunometabolic responses.

Dietary egg yolk-derived anti-interleukin (IL)-10 may preserve broiler chicken performance during coccidiosis due to Eimeria spp. infection while effects on secondary Clostridium perfringens (necrotic enteritis) are unknown. Some necrotic enteritis models implement Salmonella Typhimurium to improve repeatability; however, Salmonella upregulation of IL-10 may be a confounder when evaluating anti-IL-10. The study objective was to investigate anti-IL-10 effects on systemic cytokine concentrations and immunometabolism during E. maxima ± C. perfringens challenge in models ± S. Typhimurium. Three 25 d replicate studies using Ross 308 chicks were conducted in wire-floor cages (32 cages/ replicate) with chicks assigned to diets ± 0.03% anti-IL-10. 640 chicks (20/ cage; replicates 1 and 2) were inoculated with sterile saline ± 1×108 colony forming units (CFU) S. Typhimurium while 480 chicks (15/ cage) were placed in replicate 3. In all replicates, blood samples were collected on d 14 (6 chicks/treatment) before administering 15,000 sporulated E. maxima M6 oocysts to S. Typhimurium-inoculated (replicates 1 and 2) or challenge-designated chicks (replicate 3). Half the E. maxima-challenged chicks received 1×108 CFU C. perfringens on d 18 and 19. Blood samples were collected at 1, 3, 7, and 11 d post-inoculation (dpi) with E. maxima and 1, 3, and 7 dpi with secondary C. perfringens. Plasma cytokines were determined by ELISA while immunometabolic assays evaluated peripheral blood mononuclear cell ATP production and glycolytic rate responses. Data were analyzed with diet and challenge fixed effects plus associated interactions (SAS 9.4; P ≤ 0.05). Replicates 1 and 2 showed few immunometabolic responses within 3 dpi with E. maxima, but 25 to 31% increased ATP production and 32% increased compensatory glycolysis at 1 dpi with C. perfringens in challenged vs. unchallenged chicks (P ≤ 0.04). In replicate 3, total ATP production and compensatory glycolysis were increased 25 and 40%, respectively, by the E. maxima main effect at 1dpi (P ≤ 0.05) with unobserved responsiveness to C. perfringens. These outcomes indicate that model type had greater impacts on systemic immunity than anti-IL-10.

Broiler chicken distal jejunum microbial communities are more responsive to coccidiosis or necrotic enteritis challenge than dietary anti-interleukin-10 in a model using Salmonella Typhimurium- Eimeria maxima- Clostridium perfringens coinfection.

Dietary anti-interleukin (IL)-10 antibodies may protect broiler performance during coccidiosis by inhibiting Eimeria host-evasion pathways; however, anti-IL-10's effects on microbial communities during coccidiosis and secondary Clostridium perfringens (necrotic enteritis) challenge is unknown. The study objectives were to assess the jejunal microbiota of broilers fed anti-IL-10 during E. maxima ± C. perfringens challenge. Two replicate studies using Ross 308 chicks placed in wire-floor cages (32 cages/ replicate study; 20 chicks/ cage) were conducted, with chicks assigned to diets ± 0.03% anti-IL-10 for 25 d. In both replicate studies, challenge-designated chicks were inoculated with 1 × 108Salmonella Typhimurium colony forming units (CFU) at placement. On d14, S. Typhimurium-inoculated chicks were gavaged with 15,000 sporulated Eimeria maxima M6 oocysts and half the E. maxima-challenged chicks received 1×108C. perfringens CFUs on d 18 and 19. Six chicks/ treatment were euthanized for distal jejunum content collection at baseline (d 14), 7 d post-inoculation (pi) with E. maxima/ 3 dpi with C. perfringens (peak) or 11 dpi with E. maxima/ 7 dpi with C. perfringens (post-peak) for 16S rRNA gene amplicon sequencing. Sequences were quality screened (Mothur V.1.43.0) and clustered into de novo operation taxonomical units (OTU; 99% similarity) using the SILVA reference database (v138). Alpha diversity and log-transformed relative abundance data were analyzed in SAS 9.4 with replicate study, diet, challenge, and timepoint main effects plus associated interactions (P ≤ 0.05). Few baseline changes were observed, but E. maxima ± C. perfringens challenge reduced Romboutsia and Staphylococcus relative abundance 4- to 800-fold in both replicate studies (P ≤ 0.008). At peak challenge with secondary C. perfringens, feeding anti-IL-10 instead of the control diet reduced Clostridium sensu stricto 1 relative abundance 13- and 1,848-fold in both replicate studies (P < 0.0001); however, OTUs identified as C. perfringens were not affected by dietary anti-IL-10. These results indicate that anti-IL-10 does not affect the jejunal microbiota of unchallenged broilers, while coccidiosis or necrotic enteritis challenge generally contributed to greater microbiota alterations than diet.

Development of a TaqMan polymerase chain reaction detection method for the precise identification and quantification of an attenuated Eimeria maxima vaccine strain in poultry.

Avian coccidiosis, a parasitic disease prevalent in poultry, is caused by Eimeria species and leads to significant economic losses. The use of attenuated live oocyst vaccines has been adopted as an alternative to the use of anticoccidial drugs. However, the accurate detection and differentiation of vaccine strains from virulent ones remain challenging. Therefore, this study presents a novel TaqMan polymerase chain reaction (PCR) detection method that offers enhanced sensitivity, specificity, and reproducibility compared with traditional PCR techniques. Through whole-genome resequencing and bioinformatics analysis, we identified a molecular marker gene, Em_marker6, with a unique 21-base pair deletion specific to the Eimeria maxima attenuated vaccine strain. Optimized primers and probes targeting this marker enabled rapid quantification cycle value achievement and high fluorescence intensity. The standard curve's slope of -3.540 and correlation coefficient of 0.9971 confirmed precise quantification capabilities. The TaqMan PCR method detected as few as 30 plasmid DNA copies and 50 oocysts per reaction, outperforming traditional PCR techniques by an order of magnitude. No cross-reactivity was observed with other E. maxima wide-type strains or common intestinal pathogens, ensuring the exclusive detection of the E. maxima EMPY vaccine strain. Weekly testing over 3 weeks demonstrated minimal variability, indicating robust consistency in the method's application. Testing on 61 clinical samples revealed a 57.38% positivity rate for E. maxima species and 13.11% for the vaccine strain. The Em_marker6 gene exhibited genetic stability across multiple generations, confirming the detection method's robust stability for the attenuated E. maxima vaccine strain. This study significantly advances the field of avian coccidiosis research and control by providing a valuable tool for monitoring vaccine purity and preventing inadvertent infections in vaccinated flocks, aligning with global efforts to curb antibiotic use in animal feed.

Efficacy of a DNA vaccine carrying Eimeria maxima Gam56 antigen gene against coccidiosis in chickens.

To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscularly to yellow feathered broilers of 1-week old at 3 dosages (25, 50, and 100 µg/chick). Injection was repeated once 1 week later. One week after the second injection, birds were challenged orally with 5×10(4) sporulated oocysts of E. maxima, then weighed and killed at day 8 post challenge. Blood samples were collected and examined for specific peripheral blood lymphocyte proliferation activity and serum antibody levels. Compared with control groups, the administration of pcDNA-Gam56 vaccine markedly increased the lymphocyte proliferation activity (P<0.05) at day 7 and 14 after the first immunization. The level of lymphocyte proliferation started to decrease on day 21 after the first immunization. A similar trend was seen in specific antibody levels. Among the 3 pcDNA-Gam56 immunized groups, the median dosage group displayed the highest lymphocyte proliferation and antibody levels (P<0.05). The median dosage group had the greatest relative body weight gain (89.7%), and the greatest oocyst shedding reduction (53.7%). These results indicate that median dosage of DNA vaccine had good immunogenicity and immune protection effects, and may be used in field applications for coccidiosis control.

Evaluating a Salmonella Typhimurium, Eimeria maxima, and Clostridium perfringens coinfection necrotic enteritis model in broiler chickens: repeatability, dosing, and immune outcomes.

Coccidiosis and necrotic enteritis negatively impact poultry production, making challenge model repeatability important for evaluating mitigation strategies. Study objectives were: 1) evaluate Salmonella Typhimurium-Eimeria maxima-Clostridium perfringens necrotic enteritis coinfection model repeatability and 2) investigate E. maxima dose and early S. Typhimurium challenge on immune responses. Three trials using Ross 308 chicks assigned to 12 cages/trial (7 chicks/cage) in wire-floor brooders were completed. Trials 1 and 2 determined E. maxima dose for necrotic enteritis challenge in trial 3. Chicks in trials 1 and 2 were inoculated with 0 (control), 5, 15, or 25,000 sporulated E. maxima M6 oocysts on d 14 and jejunal lesion scores evaluated on d 20. In trial 3, chicks were assigned to control or necrotic enteritis challenge (42 chicks/group). Necrotic enteritis challenge chicks were inoculated with 1 × 105 colony forming units (CFU) S. Typhimurium on d 1, 15,000 E. maxima oocysts on d 14, and 1 × 108 CFU C. perfringens on d 19 and 20 with lesion scoring on d 22. Bird and feeder weights were recorded throughout each trial. Peripheral blood mononuclear cells (PBMC) were isolated from 1 chick/cage at baseline (all trials), 4 chicks/dose (trials 1 and 2) or 8 chicks/challenge (trial 3) 24 h post-inoculation (pi) with E. maxima for immunometabolic assays and immune cell profiling. Data were analyzed by mixed procedure (SAS 9.4) with challenge and timepoint fixed effects (P ≤ 0.05, trends 0.05 ≤ P ≤ 0.01). Inoculating chicks with 15,000 E. maxima oocysts increased d 14 to 20 FCR 79 points (trials 1 and 2; P = 0.009) vs. unchallenged chicks and achieved a target lesion score of 2. While C. perfringens challenge reduced trial 3 performance, average lesion scores were <1. Salmonella inoculation on d 1 tended to increase PBMC ATP production 41.6% 24 hpi with E. maxima vs. chicks challenged with E. maxima only (P = 0.06). These results provide insight for future model optimization and considerations regarding S. Typhimurium's effect on E. maxima immune response timelines.