Clostridium perfringens antigens and challenges for development of vaccines against necrotic enteritis in poultry.

INTRODUCTION: Chickens with Necrotic Enteritis (NE), caused by Clostridium perfringens, exhibit acute and chronic symptoms that are difficult to diagnose, leading to significant economic losses. Vaccination is the best method for controlling and preventing NE. However, only two vaccines based on the CPA and NetB toxins have been commercialized, offering partial protection, highlighting the urgent need for more effective vaccines. OBJECTIVE: This review aimed to identify promising antigens for NE vaccine formulation and discuss factors affecting their effectiveness. METHODS: A systematic review using five scientific databases identified 30 eligible studies through the Rayyan tool, which were included for quality review. RESULTS: We identified 25 promising antigens, including CPA, NetB, FBA, ZMP, CnaA, FimA, and FimB, categorized by their role in disease pathogenesis. This review discusses the biochemical, physiological, and genetic traits of recombinant antigens used in vaccine prototypes, their expression systems, and immunization potential in chickens challenged with virulent C. perfringens strains. Market supply challenges, immunogenic potential, vaccine platforms, adjuvants, and factors related to vaccination schedules-such as administration routes, dosing intervals, and age at immunization-are also addressed. Additionally, the study notes that vaccine formulations tested under mild challenges may not offer adequate field-level protection due to issues replicating aggressive conditions, strain virulence loss, and varied methodologies. CONCLUSIONS: An ideal NE vaccine should incorporate multiple antigens, molecular adjuvants, and delivery systems via in ovo and oral routes. The review underscores the challenges in developing and validating NE vaccines and the urgent need for a standardized protocol to replicate aggressive challenges accurately.

Fish gut-derived probiotic Pediococcus pentosaceus alleviates gossypol-induced intestinal inflammation by inhibiting NLRC3/NF-κB/IL-1ß signal pathway in Nile tilapia.

Cottonseed meal (CSM) and cottonseed protein concentrate (CPC) serve as protein alternatives to fish meal and soybean meal in the feed industry. However, the presence of gossypol residue in CSM and CPC can potentially trigger severe intestinal inflammation, thereby restricting the widespread utilization of these two protein sources. Probiotics are widely used to prevent or alleviate intestinal inflammation, but their efficacy in protecting fish against gossypol-induced enteritis remains uncertain. Here, the protective effect of Pediococcus pentosaceus, a strain isolated from the gut of Nile tilapia (Oreochromis niloticus), was evaluated. Three diets, control diet (CON), gossypol diet (GOS) and GOS supplemented with P. pentosaceus YC diet (GP), were used to feed Nile tilapia for 10 weeks. After the feeding trial, P. pentosaceus YC reduced the activity of myeloperoxidase (MPO) in the proximal intestine (PI) and distal intestine (DI). Following a 7-day exposure to Aeromonas hydrophila, the addition of P. pentosaceus YC was found to increase the survival rate of the fish. P. pentosaceus YC significantly inhibited the oxidative stress caused by gossypol, which was evidenced by lower reactive oxygen species (ROS) and malondialdehyde (MDA), as well as higher activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in PI and DI. Addition of P. pentosaceus YC significantly inhibited enteritis, with the lower expression of pro-inflammatory cytokines (il-1ß, il-6, il-8) and higher expression of anti-inflammatory cytokines tgf-ß. RNA-seq analysis indicated that P. pentosaceus YC supplementation significantly inhibited nlrc3 and promoted nf-κb expression in PI and DI, and the siRNA interference experiment in vivo demonstrated that intestinal inflammation was mediated by NLRC3/NF-κB/IL-1ß signaling pathway. Fecal bacteria transplantation experiment demonstrated that gut microbiota mediated the protective effect of P. pentosaceus YC. These findings offer valuable insights into the application of P. pentosaceus YC for alleviating gossypol-induced intestinal inflammation in fish.

Systematic investigation on the pharmaceutical components and mechanism of the treatment against zebrafish enteritis by Sporisorium reilianum f. sp. reilianum based on histomorphology and pathology.

ETHNOPHARMACOLOGICAL RELEVANCE: Sporisorium reilianum f. sp. reilianum (SSR) is a fungus isolated from a medicinal plant. Recorded in the "Compilation of National Chinese Herbal Medicine" and "Compendium of Materia Medica," it was used for preventing and treating intestinal diseases, enhancing immune function, etc. In this study, we investigated the chemical composition and bioactivity of SSR. Network pharmacology is utilized for predictive analysis and targeting pathway studies of anti-inflammatory bowel disease (IBD) mechanisms. Pharmacological activity against enteritis is evaluated using zebrafish (Danio rerio) as model animals. AIM OF THE STUDY: To reveal the treatment of IBD by SSR used as traditional medicine and food, based on molecular biology identification of SSR firstly, and the pharmaceutical components & its toxicities, biological activity & mechanism of SSR were explored. MATERIALS AND METHODS: Using chromatography and zebrafish IBD model induced by dextran sulfate sodium (DSS), nine compounds were first identified by nuclear magnetic resonance (NMR). The toxicity of ethanol crude extract and monomers from SSR were evaluated by evaluating the phenotypic characteristics of zebrafish embryos and larvae, histomorphology and pathology of the zebrafish model guided by network pharmacology were conducted. RESULTS: The zebrafish embryo development did not show toxicity. The molecular docking and enrichment pathway results predicted that metabolites 3 & 4 (N-trans- feruloyl-3-methoxytyramine & N-cis-feruloyl-3-methoxytyramine) and 7 & 8 (4-N- trans-p-coumaroyltyramine & 4-N-cis--p-coumaroyltyramine) have anti-enteritis activities. This paper lays an experimental foundation for developing new drugs and functional foods.

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.

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.

Supplementation with organic yeast-derived selenium provides immune protection against experimental necrotic enteritis in broiler chickens.

Necrotic enteritis (NE) is a potentially fatal poultry disease that causes enormous economic losses in the poultry industry worldwide. The study aimed to evaluate the effects of dietary organic yeast-derived selenium (Se) on immune protection against experimental necrotic enteritis (NE) in commercial broilers. Chickens were fed basal diets supplemented with different Se levels (0.25, 0.50, and 1.00 Se mg/kg). To induce NE, Clostridium perfringens (C. perfringens) was orally administered at 14 days of age post hatch. The results showed that birds fed 0.25 Se mg/kg exhibited significantly increased body weight gain compared with the non-supplemented/infected birds. There were no significant differences in gut lesions between the Se-supplemented groups and the non-supplemented group. The antibody levels against α-toxin and NetB toxin increased with the increase between 0.25 Se mg/kg and 0.50 Se mg/kg. In the jejunal scrapings and spleen, the Se-supplementation groups up-regulated the transcripts for pro-inflammatory cytokines IL-1ß, IL-6, IL-8, iNOS, and LITAF and avian ß-defensin 6, 8, and 13 (AvBD6, 8 and 13). In conclusion, supplementation with organic yeast-derived Se alleviates the negative consequences and provides beneficial protection against experimental NE.

Research Note: Clostridium perfringens NetB and CnaA neutralizing nanobodies in feed reduce the incidence of poultry necrotic enteritis.

Necrotic enteritis is a devastating disease to poultry caused by the bacterium Clostridium perfringens. As a novel approach to combating poultry necrotic enteritis, we identified and characterized several hundred single domain antibody fragments (or nanobodies) capable of binding either the NetB toxin or the collagen-binding adhesin (CnaA) of C. perfringens. Many of the nanobodies could neutralize the in vitro functions of NetB or CnaA with inhibitory concentrations in the nanomolar range. The nanobodies were also screened for proteolytic stability in an extract derived from gastrointestinal tract fluids of chickens. A collection of 6 nanobodies (4 targeting NetB and 2 targeting CnaA) with high neutralizing activity and high gastrointestinal tract extract stability were expressed and secreted by Pichia pastoris or Bacillus subtilis. Chickens were given a feed with 1 of the 2 nanobody-containing groups: 1) nanobody-containing P. pastoris supernatants that were semi-purified, lyophilized, and enterically coated, or 2) B. subtilis spores from strains containing the nanobody genes. Compared to untreated chickens (23.75% mortality), mortality of chickens receiving feed modified with the P. pastoris and B. subtilis products decreased to 11.25 and 7.5%, respectively. These results offer a new opportunity to improve the control of poultry necrotic enteritis by incorporating highly specific nanobodies or bacteria expressing these nanobodies directly into chicken feed.

Bromelain can reduce the negative effects of a subclinical necrotic enteritis in broiler chickens.

This study was conducted to examine the efficacy of a bromelain-based supplementation coded ANR-pf on growth performance and intestinal lesion of broiler chickens under necrotic enteritis (NE) challenge. A total of 540 Ross 308 day-old male chicks were randomly allocated into 6 treatments of 6 replicates. The bromelain formulation was delivered to chickens through gavaging or in drinking water method twice, on d 8 and 13. Nonchallenged groups included 1) without or 2) with the specific bromelain formulation gavaged at 0.8 mL/kg. NE-challenged groups included 3) without the specific bromelain formulation; 4) gavaged with 0.4 mL/kg; 5) gavaged with 0.8 mL/kg and 6) supplemented with 0.8 mL/kg via drinking water. Birds were challenged with Eimeria spp. on d 9 and Clostridium perfringens (NE-18 strain) on d 14 and 15. On d 14 and 19, fresh faecal contents were collected for the determination of oocyst counts. Intestinal lesion scores were determined on d16. Performance and mortality were recorded throughout the entire experiment. Among challenged groups, birds received additive via drinking water had higher weight gain (WG) compared to the remaining groups (P < 0.001) in the grower phase and had lower FCR compared to 0.4 mL/kg inoculated group in the grower and finisher phases (P < 0.001). Bromelain supplementation via drinking water improved the WG of challenged birds, similar to that of the nonchallenged birds (P < 0.001), and lowered FCR compared to other challenged groups (P < 0.001). Nonchallenged birds and birds that received bromelain formulation in drinking water did not have lesions throughout the small intestine whereas challenged birds, either un-supplemented or supplemented with bromelain via inoculation route recorded similar lesion score levels in the jejunum. At d 19, birds received bromelain in drinking water had lower fecal oocyst numbers compared to challenged birds without additive (P < 0.001). In conclusion, bromelain administration via drinking water could ameliorate the negative impacts of NE-infection in broilers by improving performance, lowering the oocyst numbers and lesion scores.

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.

Chlorogenic acid protects against intestinal inflammation and injury by inactivating the mtDNA-cGAS-STING signaling pathway in broilers under necrotic enteritis challenge.

This study aimed to determine the effects of chlorogenic acid (CGA) on the growth performance, intestinal health, immune response, and mitochondrial DNA (mtDNA)-cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway in broilers under necrotic enteritis (NE) challenge. The 180 one-day-old male Cobb 500 broilers with similar body weight of 44.59 ± 1.39 g were randomly allocated into 3 groups. The groups were control diet (Control group), control diet + NE challenge (NE group), and control diet + 500 mg/kg CGA + NE challenge (NE + CGA group), with 6 replicates per treatment. All broilers except the Control group were given sporulated coccidian oocysts (d 14) and Clostridium perfringens (d 19-21) by oral gavage. Our findings showed that CGA improved the growth performance and intestinal morphology in broilers under NE challenge. CGA supplementation elevated the barrier function in broilers under NE challenge, which reflected in the decreased serum concentrations of D-lactate and diamine oxidase, and upregulated jejunal protein expression of occludin. CGA supplementation also improved the immune function, which reflected in the increased concentrations and gene expressions of anti-inflammatory factors, and decreased concentrations and gene expressions of proinflammatory factors. CGA supplementation further enhanced intestinal cell proliferation and differentiation, which manifested in the increased number of goblet cells and positive cells of proliferating cell nuclear antigen on d 28 and 42. Furthermore, CGA supplementation decreased the mtDNA (d 42) and mitochondrial reactive oxygen species levels (d 28 and 42), and increased the mitochondrial membrane potential (d 42) and mitochondrial complex I (d 28 and 42) or III (d 28) activity. Broilers challenged with NE had upregulated jejunal protein expressions of cGAS, phospho-TANK-binding kinase 1, and phospho-interferon regulatory factor 7 compared with the Control group, which were downregulated after CGA supplementation. In conclusion, dietary supplementation CGA could protect against intestinal inflammation and injury by reducing the leakage of mtDNA and inactivating the cGAS-STING signaling pathway in broilers under NE challenge.

Molecular and cellular characterization of immunity conferred by lactobacilli against necrotic enteritis in chickens.

Necrotic enteritis is an important enteric disease of poultry that can be controlled with in-feed antibiotics. However, with the concerns over antimicrobial resistance, there is an increased interest in the use of alternatives. Probiotics are one of the alternatives that have gained considerable attention due to their antimicrobial and immunomodulatory activities. Therefore, in the present study, we evaluated the effects of two different Lactobacillus species alone or as a cocktail on prevention of necrotic enteritis. Day-old male broiler chickens were divided into five groups and on days 1, 8, 15, and 22, birds in groups 2 and 3 received 1×108 colony forming units (CFU) of L. johnsonii and L. reuteri, respectively. Group 4 received probiotic cocktails containing both bacteria (108 CFU/bird) and the negative and positive control groups did not receive any lactobacilli. Starting on day 23 post-hatch, birds in all groups (except the negative control group) were orally challenged twice per day with 3×108 CFU of a pathogenic C. perfringens strain for 3 days. Tissue and cecal samples were collected before and after challenge to assess gene expression, lymphocyte subsets determination, and microbiome analysis. On day 26 of age, lesion scoring was performed. The results demonstrated that the group that received the lactobacilli cocktail had significantly reduced lesion scores compared to the positive control group. In addition, the expression of interleukin (IL)-12 in the jejunum and CXC motif chemokine ligand 8 (CXCL8), IL-13, and IL-17 in the ileum were downregulated in the group that received the lactobacilli cocktail when compared to the positive control. Treating chickens with the lactobacilli cocktail prior to challenge enhanced the percentage of CD3-CD8+ cells and Bu-1+IgY+ B cells in the ileum and increased the frequency of monocyte/macrophages, CD3-CD8+ cells, Bu-1+IgM+, and Bu-1+IgY+ B cells in the jejunum. Treatment with the lactobacilli cocktail reduced the relative expression of Gamma-Protobacteria and Firmicutes compared to the positive control group. In conclusion, the results presented here suggest that treatment with the lactobacilli cocktail containing L. johnsonii and L. reuteri reduced necrotic enteritis lesions in the small intestine of chickens, possibly through the modulation of immune responses.

Evaluation of Three Vaccination Schemes Against Clostridium perfringens Alpha Toxin and Their Effects on the Performance, Level of Intestinal Lesions, and Serum Antibody Titers in Broilers.

The objective of the trial was to evaluate three vaccination schemes against Clostridium perfringens (CP) alpha-toxoid through drinking water to determine if they can protect against clinical signs of necrotic enteritis and coccidiosis in broiler chickens. Three hundred 1-day-old Cobb 500 male chicks were used in 4 treatments with 10 repetitions. Each group received 1 of the following treatments over the course of 29 days: T1, no vaccination; T2, vaccination on Day 1; T3, vaccination on Day 7; and T4, vaccination on Days 7 and 17. The birds were vaccinated with inactivated CP toxoid type A, administered via drinking water. During the first 14 days, a high-protein diet (27%) consisting of corn, soy, and fish meal was fed. On Day 14 Eimeria acervulina (EA), Eimeria maxima (EMx), Eimeria tenella (ET), Eimeria necatrix, and Eimeria brunetti were used in a coccidial challenge. The field isolate CP type A was then inoculated on Days 18, 19, and 20. Ten birds were slaughtered by treatment to obtain serology samples for antibody titers and intestine samples for CP and Eimeria lesion score and gut integrity indicators. Productive performance was assessed using complete randomized design and compared statistically using the Tukey test, whereas intestinal integrity variables and antibodies against CP alpha toxin were assessed using a Kruskal-Wallis nonparametric method. The results revealed that the treatments had an effect on productive performance (P < 0.05); T3 had better body weight and weight gain than T1. In terms of lesion score at Day 21, T4 had a lower lesion score by EA, EMx, and ET than T1. Cell desquamation in T2 was lower than in T4, and excess mucus (EM) in T1 was the worst in gut integrity indicators at Day 21. On the other hand, T2 had more EM than T3 and T4 at Day 25. In the measurement of antibodies, no statistical differences (P > 0.05) were found. These findings indicate that vaccination on Day 7 (T3) outperformed double vaccination on Days 7 and 17 (T4) and single on Day 1 (T2), in terms of productive performance, gut integrity, and lesion scores; and on the last day of the experiment T3 had the best performance in immunology response.

Evaluación de tres esquemas de vacunación contra la toxina alfa de Clostridium perfringens y sus efectos sobre el rendimiento, el nivel de lesiones intestinales y los títulos de anticuerpos séricos en pollos de engorde. El objetivo del ensayo fue evaluar tres programas de vacunación contra Clostridium perfringens (CP) con un alfa-toxoide a través del agua de bebida para determinar si protegían contra signos clínicos de enteritis necrótica y coccidiosis en pollos de engorde. Para ello se emplearon 300 pollitos machos Cobb 500 de un día de edad, distribuidos en 4 tratamientos con 10 repeticiones. Cada grupo recibió, durante 29 días, uno de los siguientes tratamientos: T1: sin vacunación; T2: vacunación en el día uno; T3: vacunación en el día siete y T4, vacunación en los días siete y 17. Las aves fueron vacunadas con toxoide inactivado de C. perfringens tipo A, que se administró en el agua de bebida. Durante los primeros 14 días se alimentó con una dieta alta en proteína (27%) que consistía en maíz, soya y harina de pescado. El desafío coccidial se realizó en el día 14 con Eimeria acervulina (EA), Eimeria maxima (EMx), Eimeria tenella (ET), Eimeria necatrix and Eimeria brunetti. Posteriormente, en los días 18, 19 y 20 se inoculó una cepa aislada de campo de C. perfringens tipo A. Se sacrificaron diez aves por tratamiento para obtener muestras de sueros para determinar los títulos de anticuerpos y muestras de intestino para determinar la puntuación de lesiones por C. perfringens, por Eimeria y los indicadores de integridad intestinal. El comportamiento productivo se analizó bajo un diseño completamente al azar (DCA) y la comparación estadística se realizó mediante la prueba de Tukey, mientras que para las variables de integridad intestinal y los títulos de anticuerpos contra alfa toxina de C. perfringens se utilizó el método no paramétrico Kruskal-Wallis. Los resultados mostraron que el comportamiento productivo fue influenciado por los tratamientos (P < 0.05); el tratamiento T3 mostró el mejor peso corporal y ganancia de peso en comparación con el tratamiento T1. Con relación al puntaje de lesiones en el día 21, el tratamiento T4 tuvo el menor puntaje de lesiones por E. acervulina, E. maxima y E. tenella en comparación con el tratamiento T1. La descamación celular en el tratamiento T2 fue menor que en el T4 y el exceso de moco en el tratamiento T1 fue peor entre los indicadores de integridad intestinal en el día 21. Por otro lado, el tratamiento T2 tenía más exceso de moco en comparación con los tratamientos T3 y T4 en el día 25. No se encontraron diferencias estadísticas (P > 0.05) en la medición de títulos de anticuerpos. Estos hallazgos indican que la vacunación en el día siete (T3) superó a la vacunación doble en los días 7 y 17 (T4) y única en el día uno (T2), en términos de rendimiento productivo, integridad intestinal y puntajes de lesiones, además en el último día del experimento, el tratamiento T3 tuvo el mejor desempeño en la respuesta inmunológica.

Hyperimmune egg yolk antibodies developed against Clostridium perfringens antigens protect against necrotic enteritis.

Necrotic enteritis (NE) is a widespread infectious disease caused by Clostridium perfringens that inflicts major economic losses on the global poultry industry. Due to regulations on antibiotic use in poultry production, there is an urgent need for alternative strategies to mitigate the negative effects of NE. This paper presents a passive immunization technology that utilizes hyperimmune egg yolk immunoglobulin Y (IgY) specific to the major immunodominant antigens of C. perfringens. Egg yolk IgYs were generated by immunizing hens with 4 different recombinant C. perfringens antigens, and their protective effects against NE were evaluated in commercial broilers. Six different spray-dried egg powders were produced using recombinant C. perfringens antigens: α-toxin, NE B-like toxin (NetB; EB), elongation factor-Tu (ET), pyruvate:ferredoxin oxidoreductase, a mixture of 4 antigens (EM-1), and a nonimmunized control (EC). The challenged groups were either provided with different egg powders at a 1% level or no egg powders (EN). The NE challenge model based on Eimeria maxima and C. perfringens dual infection was used. In Experiments 1 and 2, the EB and ET groups exhibited increased body weight gain (BWG; P < 0.01), decreased NE lesion scores (P < 0.001), and reduced serum NetB levels (P < 0.01) compared to the EN and EC groups. IgY against NetB significantly reduced Leghorn male hepatocellular cytotoxicity in an in vitro test (P < 0.01). In Experiment 3, the protective effect of the IgYs mixture (EM-2) against C. perfringens antigens (NetB and EFTu) and Eimeria antigens (elongation factor-1-alpha: EF1α and Eimeria profilin: 3-1E) was tested. The EM-2 group showed similar body weight, BWG, and feed intake from d 7 to 22 compared to the NC group (P < 0.05). On d 20, the EM-2 group showed comparable intestinal permeability, NE lesion scores, and jejunal NetB and collagen adhesion protein levels to the NC group (P < 0.05). In conclusion, dietary mixture containing antibodies to NetB and EFTu provides protection against experimental NE in chickens through passive immunization.

A Recombinant Duck Plague Virus Containing the ICP27 Deletion Marker Provides Robust Protection in Ducks.

Duck plague virus (DPV) is a member of Alphaherpesvirus genus and poses a major threat to waterfowl breeding. Genetic engineered vaccines that are capable of distinguishing naturally infected from vaccine-immunized animals are useful for eradicating duck plague. In this study, reverse genetics was used to develop an ICP27-deficient strain (CHv-ΔICP27), and its potential as a marker vaccination candidate was evaluated. The results showed that the CHv-ΔICP27 generated in this study exhibited good genetic stability in vitro and was highly attenuated both in vivo and in vitro. The level of neutralizing antibody generated by CHv-ΔICP27 was comparable to that induced by a commercial DPV vaccine, suggesting that it could protect ducks from virulent DPV attack. By using molecular identification techniques such as PCR, restriction fragment length polymorphism, immunofluorescence, Western blotting, and others, it is possible to differentiate the CHv-ΔICP27 from wild-type strains. Moreover, ICP27 can also be a potential target for the genetic engineering vaccine development of alphavirus or perhaps the entire herpesvirus family members due to the highly conservative of ICP27 protein in all herpesvirus family members. IMPORTANCE The development of distinguishable marker vaccines from natural infection is a key step toward eradicating duck plague. Here, we generated a recombinant DPV that carries an ICP27 deletion marker that could be easily distinguished from wild-type strain by molecular biological methods. It was highly attenuated in vitro and in vivo and could provide comparable protection to ducks after a single dose of immunizations, as commercial vaccines did. Our findings support the use of the ICP27-deficient virus as a marker vaccine for DPV control and future eradication.

The protective effects of Bacillus licheniformis against inflammatory responses and intestinal barrier damage in broilers with necrotic enteritis induced by Clostridium perfringens.

BACKGROUND: Bacillus licheniformis is a gram-positive bacterium that has strong environmental adaptability and can improve the growth performance, immunity, and antioxidant function of broilers. The current study aimed to elucidate the protective capability of B. licheniformis against inflammatory responses and intestinal barrier damage in broilers with necrotic enteritis (NE) induced by Clostridium perfringens (CP). RESULTS: The results showed that B. licheniformis enhanced the final body weight in broilers compared with that of broilers in the CP group after the stress of infection (P < 0.05). Bacillus licheniformis reversed the decreased levels of serum and jejunum mucosa immunoglobulins and anti-inflammatory cytokines, reduced the values of villus height and the ratio of villus height to crypt depth, and mitigated the increased levels of serum d-lactic acid and diamine oxidase in CP-challenged broilers (P < 0.05). Moreover, B. licheniformis modulated the expression levels of genes involved in the TLR4/NF-κB signalling pathway, the NLRP3 inflammasome activation pathway, and the sirt 1/Parkin signalling pathway in CP-challenged broilers. Compared with the CP challenge group, the B. licheniformis-treated group exhibited reduced abundance values of Shuttleworthia and Alistipes and enhanced abundance values of Parabacteroides in the caecal contents (P < 0.05). CONCLUSION: Bacillus licheniformis improved the final body weight and alleviated the inflammatory response and intestinal barrier function damage in birds with NE induced by CP by maintaining intestinal physiological function, enhancing immunity, regulating inflammatory cytokine secretion, modulating the mitophagy response, and increasing the abundance of beneficial intestinal flora. © 2023 Society of Chemical Industry.

Efficacy of Antibiotic and Non-antibiotic Interventions in Preventing and Treating Necrotic Enteritis in Broiler Chickens: A Systematic Review.

The objective of this systematic review was to compare the efficacy of antibiotic and non-antibiotic alternatives in the prevention and treatment of necrotic enteritis (NE) in broiler chickens. In vivo experimental and observational studies that compared the administration of non-antibiotic compounds with antibiotics to prevent or treat NE in broiler chickens and that evaluated mortality and/or clinical or subclinical NE outcome measures were eligible. Four electronic databases were searched in December 2019 and updated in October 2021. Retrieved studies were evaluated in two phases: abstract and design screening. Data were then extracted from included studies. Risk of bias was assessed by outcome following the Cochrane Risk of Bias 2.0 tool. A meta-analysis was not conducted due to heterogeneity across interventions and outcomes. The non-antibiotic and antibiotic groups were compared at the outcome level for individual studies using the mean difference and 95% confidence interval (CI) calculated post hoc from raw data. In total, 1282 studies were originally identified, and 40 were included in the final review. The overall risk of bias for the 89 outcomes was either "high" (n = 34) or "some concerns" (n = 55). Individual study comparisons showed a beneficial trend toward the antibiotic group for reduced mortality, NE lesion scores (overall, jejunum, and ileum), Clostridium perfringens counts, and for most histologic measurements (duodenum, jejunum, and ileum villi height, and jejunum and ileum crypt depth). The non-antibiotic groups showed a beneficial trend for NE duodenum lesion scores and duodenum crypt depth measurements. Based on this review, there is a trend that mostly favors antibiotic compounds in preventing and/or treating NE, but the evidence also suggests no difference when comparing them with non-antibiotic alternatives. Studies assessing this research question were heterogeneous in their intervention conditions and outcomes measured, and there were key aspects of the experimental design not reported in some of the studies.

Eficacia de las intervenciones con antibióticos y compuestos no antibióticos para prevenir y tratar la enteritis necrótica en pollos de engorde: Una revisión sistemática El objetivo de esta revisión sistemática fue comparar la eficacia de antibióticos y alternativas a los antibióticos en la prevención y tratamiento de la enteritis necrótica (NE) en pollos de engorde. Se incluyeron estudios experimentales in vivo y estudios observacionales que compararon la administración de compuestos no considerados antibióticos con compuestos antibióticos usados para prevenir o tratar la enteritis necrótica en pollos de engorde, y que evaluaran mortalidad, signos clínicos, u otros resultados subclínicos. Se buscaron referencias en cuatro bases de datos bibliográficos en Diciembre de 2019 y por segunda vez en Octubre de 2021. Los estudios que se encontraron se evaluaron en dos fases: resumen y diseño del estudio de escrutinio. Posteriormente se extrajeron los datos de aquellos estudios que se incluyeron después del escrutinio. Se evaluó el riesgo de sesgos siguiendo la herramienta de Cochrane Risk of Bias 2.0. No se pudo realizar un meta-análisis debido a la heterogeneidad de las intervenciones y de los resultados de los estudios incluidos. Los grupos de compuestos no antibióticos y de antibióticos se compararon con base a los resultados individuales de cada estudio usando la diferencia entre medias e intervalos de confianza al 95%, calculados post-hoc usando los datos originales. Se identificaron un total de 1282 estudios, y 40 fueron finalmente incluidos en la revisión. El riesgo de sesgos para el total de los 89 resultados individuales fue alto (n = 34) o con "ciertos problemas" (n = 55). Las comparaciones individuales entre estudios mostraron una tendencia beneficiosa hacia el grupo de antibióticos en términos de mortalidad reducida, puntaje de lesiones de enteritis necrótica (total, yeyuno e íleo), conteos de Clostridium perfringens, así como para la mayoría de las medidas histológicas (altura de las vellosidades del duodeno, yeyuno e íleo, y profundidad de la cripta del yeyuno e íleo). El grupo de no antibióticos mostró una tendencia beneficiosa para el puntaje de lesiones de enteritis necrótica del duodeno y para las medidas de profundidad de la cripta del duodeno. Según esta revisión, hay una tendencia que favorece al grupo de antibióticos en la prevención y/o tratamiento de enteritis necrótica, pero la evidencia también sugiere que no hay diferencia entre los grupos. Los estudios incluidos en la comparación eran muy heterogéneos en cuanto a las condiciones de las intervenciones y a los resultados que se midieron, además de que algunos aspectos importantes del diseño experimental en algunos de los estudios no se reportaron.

Spray-dried porcine plasma enhances feed efficiency, intestinal integrity, and immune response of broilers challenged with necrotic enteritis.

Re-emergence of enteric diseases in the postantibiotic era has imposed severe loss to the poultry industry leading to the urgent need for appropriate additives to maintain gut health. Recently, more attention has been paid to animal plasma due to its high concentrations of active components such as albumins and globulins. The objective of this study was to evaluate the effects of spray-dried porcine plasma (SDP) supplementation during the starter phase (d 0-10) on growth performance, intestine health, and immune response of broilers under necrotic enteritis (NE) challenge. A total of 720 day-old male broiler parental line chicks (Ross 308) were randomly assigned to a 2 (NE challenge: no, yes) × 2 (SDP: 0, 2%) factorial arrangement with 12 replications of 15 chicks each. To induce NE, birds were inoculated with live Eimeria vaccine on d 9 and Clostridium perfringens on d 14. The body weight of birds and feed consumption were measured per pen on d 8, 10, 24, and 29 to calculate performance parameters. On d 16, three birds per pen were sampled to analyse the intestinal lesion score, gut permeability, villi morphology, relative weight of organs, and immune response. Results showed that SDP improved (P < 0.001) FCR in the pre-challenge phase (d 0-8). The results indicated that supplementing SDP lowered (P < 0.01) FCR at the end of the experiment (d 29). Dietary SDP decreased (P < 0.05) the concentration of FITC-d in serum samples of challenged broilers, although it did not affect the intestinal morphology and lesion score. Birds fed with SDP had a higher (P < 0.05) relative weight of bursa (g/kg live body weight) compared to non-supplemented birds. Supplementing SDP reduced the concentration of interleukin-6 (P < 0.05) and α-1 acid glycoprotein (P = 0.051) in serum samples of broilers. In conclusion, supplementation of SDP in the starter phase enhanced feed efficiency and gut integrity in NE challenged broilers, possibly through manipulating the immune response, while further studies targeting intestinal microflora and key genes are required to explore the mode of action.

Compound Probiotics Improve Body Growth Performance by Enhancing Intestinal Development of Broilers with Subclinical Necrotic Enteritis.

The aim of this study is to explore whether or not the combined application of BS15 and H2 is capable to have a more effective control effect on SNE in broilers. A total of 240 1-day-old female chickens were randomly divided into 5 groups: (a) basal diet in negative control group (NC group); (b) basal diet + SNE infection (coccidiosis vaccine + CP) (PC group); (c) basal diet + SNE infection + H2 pre-treatment (BT group); (d) basal diet + SNE infection + BS15 pre-treatment (LT group); and (e) basal diet + SNE infection + H2 pre-treatment + BS15 pre-treatment (MT group). The results showed the MT group had the most positive effect on inhibiting the negative effect of growth performance at 42 days of age. In the detection of the NC, PC, and MT group indicators at 28 days of age, we found that MT group significantly promoted ileum tissue development of broilers, and the ileum of broilers in the MT group formed a flora structure different from NC and PC, although it was found that the MT group had no effect on the butyrate level in the cecum, but it could affect the serum immune level, such as significantly reducing the level of pro-inflammatory cytokine IL-8 and increasing the content of immunoglobulin IgM and IgG. In conclusion, the composite preparation of Lactobacillus johnsonii BS15 and Bacillus licheniformis H2 could effectively improve the growth performance against SNE broilers, which is possibly caused by the improvement of the immune levels, the reduction of inflammation levels, and the promotion of the intestinal development.

The in vitro effect of lactose on Clostridium perfringens alpha toxin production and the implications of lactose consumption for in vivo anti-alpha toxin antibody production.

Necro-hemorrhagic enteritis in calves, caused by Clostridium perfringens type A, is a fatal disease, mostly affecting calves in intensive rearing systems. The lack of development of active immunity against α toxin, an essential virulence factor in the pathogenesis, has been proposed as a main trigger. In this experimental study, the effect of a set of milk replacer components on α toxin production, and the effect of lactose on in vivo antibody production, were investigated. For the latter, Holstein-Friesian bull calves (n = 18) were fed an all liquid diet that contained either a milk replacer with high-lactose content (45% DM) or the same milk replacer that was lactase treated, resulting in a lactose-free equivalent. Antibody levels against α toxin were monitored from 2 to 12 wk of age. In the in vitro part of the study, a concentration-dependent inhibitory effect of lactose on in vitro C. perfringens α toxin activity was observed, whereas protein did not influence α toxin activity. The in vivo experiment then showed from the age of 10 wk onwards, that anti-α toxin antibody levels of high-lactose animals declined, whereas antibody levels of the animals consuming lactose-free milk replacer remained the same throughout the trial. This points to a natural decline in maternal immunity of lactose-consuming animals, that is not compensated by the development of an active immunity, resulting in inferior protection. This study suggests that dietary lactose reduces C. perfringens α toxin production in vivo, which may lead to a decreased antigen presentation and thus lower serum antibody levels against the toxin. Consequently, any event causing massive α toxin production puts lactose-consuming calves at higher risk of developing necro-hemorrhagic enteritis.