Comparison of necrotic enteritis effects on growth performance and intestinal health in two different meat-type chicken strains Athens Canadian Random Bred and Cobb 500.

Chickens have undergone genetic improvements in the past few decades to maximize growth efficiency. However, necrotic enteritis (NE), an enteric disease primarily caused by C. perfringens, remains a significant problem in poultry production. A study investigated the differences in intestinal health between the nonselected meat-type chicken Athens Canadian Random Bred (ACRB) and the modern meat-type Cobb 500 broilers (Cobb) when challenged with experimental NE. The study utilized a 2 × 3 factorial arrangement, consisting of two main effects of chicken strain and NE challenge model (nonchallenged control, NC; NE challenge with 2,500/12,500 Eimeria maxima oocysts + 1 × 109C. perfringens, NE2.5/NE12.5). A total of 432 fourteen-day-old male ACRB and Cobb were used until 22 d (8 d postinoculation with E. maxima on d 14, dpi), and the chickens were euthanized on 6 and 8 dpi for the analysis. All data were statistically analyzed using a two-way ANOVA, and Student's t-test or Tukey's HSD test was applied when P < 0.05. The NE12.5 group showed significant decreases in growth performance and relative growth performance from d 14 to 20, regardless of chicken strain (P < 0.01). The ACRB group exhibited significant decreases in relative body weight and relative body weight gain compared to the Cobb group from d 14 to 22 (P < 0.01). On 6 and 8 dpi, both NE challenge groups showed significant decreases in intestinal villus height to crypt depth ratio, jejunal goblet cell count, and jejunal MUC2 and LEAP2 expression (P < 0.01). Additionally, the NE12.5 group had significantly higher intestinal NE lesion score, intestinal permeability, fecal E. maxima oocyst count, intestinal C. perfringens count, and jejunal IFNγ and CCL4 expression compared to the NC group (P < 0.05). In conclusion, NE negatively impacts growth performance and intestinal health in broilers, parameters regardless of the strain.

Validating the use of a newly developed cinnamaldehyde product in commercial broiler production.

Essential oils (EOs) have been considered as an alternative to antibiotics for animal production. In the current study, 4 trials were conducted on a commercial broiler farm to investigate the effects of dietary supplementation of an encapsulated cinnamon EO product (NE-OFF) on the bird growth performance, gut health, and gene expression in the ileum, spleen, and liver relating to the host response to heat and other stresses, including potential NE challenge. In each trial, approximately 30,000 Cobb or Ross broilers were randomly allocated to 4 treatments: a raised without antibiotics (RWA) commercial diet as positive control, an adjusted RWA commercial diet as negative control, and the negative control diet supplemented with 2 different dosages of NE-OFF, which was added during feed pelleting. Although the final average body weight did not differ significantly among treatment groups, birds fed NE-OFF had an increased ratio of villus height and crypt depth in the jejunum, and reduced fecal oocyst counts. Trial 2 was conducted in the summer and had a necrotic enteritis (NE) outbreak. The supplementation of NE-OFF reduced the NE incidence and bird mortality. The samples from Trial 2 were hence selected for the analyses of Clostridium perfringens and NetB toxin gene abundance in the ileum, and host responses. The C. perfringens population appeared to be positively correlated with the NetB gene abundance. The gene expression analysis suggested that NE-OFF supplementation improved nutrient absorption and transportation as well as antioxidant activities to help the birds against stress. These on-farm trial results support the hypothesis that the use of NE-OFF as a feed additive can improve bird gut health and performance in commercial broiler production, especially for preventing NE outbreaks when birds are under stress.

Natural intraepithelial lymphocyte populations rise during necrotic enteritis in chickens.

Intraepithelial lymphocytes (IEL) reside in the epithelium at the interface between the contents of the intestinal lumen and the sterile environment of the lamina propria. Because of this strategic location, IEL play a crucial role in various immunological processes, ranging from pathogen control to tissue stability. In mice and humans, IEL exhibit high diversity, categorized into induced IEL (conventional CD4 and CD8αß T cells) and natural IEL (TCRαßCD8αα, TCRγδ, and TCRneg IEL). In chickens, however, the subpopulations of IEL and their functions in enteric diseases remain unclear. Thus, we conducted this study to investigate the role of IEL populations during necrotic enteritis (NE) in chickens. At 14 days of age, sixty-three Specific-pathogen-free (SPF) birds were randomly assigned to three treatments: Control (sham challenge), Eimeria maxima challenge (EM), and Eimeria maxima + Clostridium Perfringens (C. Perfringens) co-challenge (EM/CP). The EM and EM/CP birds were infected with Eimeria maxima at day 14 of age, and EM/CP birds were additionally orally inoculated with C. perfringens at days 18 and 19 of age. Birds were weighed at days 18, 20, and 26 of age to assess body weight gain (BWG). At 20 days of age (1 day-post C. perfringens infection; dpi), and 26 days of age (7 dpi), 7 birds per treatment were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. The EM/CP birds exhibited subclinical NE disease, lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The EM/CP group showed substantial increases in the total number of natural IEL subsets, including TCRαß+CD4-CD8-, TCRαß+CD8αα+, TCRγδ+, TCRneg and innate CD8α (iCD8α) cells by at least two-fold. However, by 7 dpi, only the number of TCRαß+CD4-CD8- and TCRαß+CD8αα+ IEL maintained their increase in the EM/CP group. The EM/CP group had significantly higher expression of proinflammatory cytokines (IL-1ß and IFN-γ) and Osteopontin (OPN) in the jejunum at 1 dpi. These findings suggest that natural IEL with innate and innate-like functions might play a critical role in the host response during subclinical NE, potentially conferring protection against C. perfringens infection.

Treatment of chickens with lactobacilli prior to challenge with Clostridium perfringens modifies innate responses and gut morphology.

Necrotic enteritis caused by Clostridium perfringens (CP), is a common enteric disease of poultry that has been previously controlled by in-feed antibiotics. However, due to the rapid emergence of antimicrobial resistance, alternatives to antibiotics such as probiotics have received considerable attention because of their immunomodulatory and intestinal health benefits. The present study investigated the effects of probiotic lactobacilli on gut histomorphology and intestinal innate responses in chickens. Day-old male broiler chickens were treated with 1 × 107 or 1 × 108 colony-forming units (CFU) of a lactobacilli cocktail on days 1, 7, 14, and 20 post-hatch, while control groups were not treated with lactobacilli. On day 21, birds in all groups (except the negative control) were challenged with 3 × 108 CFU of CP for 3 days. Intestinal tissue samples were collected before and after the CP challenge to assess gene expression and for histomorphological analysis. Lactobacilli treatment at a dose of 1 × 108 CFU conferred partial protection against NE by lowering lesion scores, increasing villus height in the ileum and reducing crypt depth in the jejunum. In addition, 1 × 108 CFU of lactobacilli enhanced the expression of Toll-like receptor (TLR) 2, interferon-gamma (IFN-γ), interleukin (IL)-10, IL-12, and IL-13 in both the jejunum and ileum at different timepoints and subsequently decreased the expression of transforming growth factor beta (TGF-ß) and IL-1ß post-CP challenge. In conclusion, the results indicate that treatment with lactobacilli mitigated NE in a dose-dependent manner via improvement of intestinal morphology and modulation of innate immune response in chickens.

Bacillus subtilis HW2 enhances growth performance and alleviates gut injury via attenuation of endoplasmic reticulum stress and regulation of gut microbiota in broilers under necrotic enteritis challenge.

This study investigated the effects of Bacillus subtilis HW2 on the growth performance, immune response, endoplasmic reticulum (ER) stress, and intestinal health in broilers with necrotic enteritis. Three hundred 1-day-old male Cobb 500 broilers (33.88 ± 2.34 g) were randomly allocated to 5 groups including non-infected control (NC group), basal diet + necrotic enteritis challenge (NE group), basal diet + 1 × 106 CFU/g B. subtilis HW2 + necrotic enteritis challenge (L-Pro group), basal diet + 5 × 106 CFU/g B. subtilis HW2 + necrotic enteritis challenge (M-Pro group), and basal diet + 1 × 107 CFU/g B. subtilis HW2 + necrotic enteritis challenge (H-Pro group), with 6 replicates per group. All broilers except NC group were orally given with sporulated coccidian oocysts at day 14 and Clostridium perfringens from days 19 to 21. Results showed that L-Pro and M-Pro groups improved growth performance and intestinal morphology in necrotic enteritis-challenged broilers, and L-Pro, M-Pro, and H-Pro groups improved intestinal barrier function and immune response and decreased ER stress in necrotic enteritis-challenged broilers. Analysis of the gut microbiota revealed that L-Pro group increased the abundances of Alistipes, Coprobacter, Barnesiella, and Limosilactobacillus, decreased Erysipelatoclostridium abundance on day 42 in necrotic enteritis-challenged broilers. M-Pro group increased Turicibacter abundance on day 28 and the abundances of Alistipes, Barnesiella, and Limosilactobacillus on day 42 in necrotic enteritis-challenged broilers. H-Pro group decreased Romboutsia abundance on day 28 and unidentified_Clostridia abundance on day 42 in necrotic enteritis-challenged broilers. Analysis of short-chain fatty acids (SCFAs) revealed higher isobutyric acid and isovaleric acid levels in L-Pro and M-Pro groups than NE group. Correlation analysis revealed the correlations between the biochemical parameters and gut microbiota as well as SCFAs, especially Romboutsia, Barnesiella, Coprobacter, isobutyric acid, and isovaleric acid. Overall, our results indicated that B. subtilis HW2 supplementation could ameliorate necrotic enteritis infection-induced gut injury. The optimal dietary supplementation dosage of Bacillus subtilis HW2 was 5 × 106 CFU/g.

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.

A microbial muramidase improves growth performance and reduces inflammatory cell infiltration in the intestine of broilers chickens under Eimeria and Clostridium perfringens challenge.

The objective of the present studies was to evaluate muramidase (MUR) supplementation in broilers under Eimeria and/or Clostridium perfringens challenge. For this, 2 experiments were conducted. Experiment 1. A total of 256 one-day old male Cobb 500 chicks were placed in battery cages in a completely randomized design, with 5 treatment groups, 7 replicate cages per treatment and 8 birds per cage. The treatments were: nonchallenged control (NC), challenged control (CC), CC + MUR at 25,000 or 35,000 LSU(F)/kg, and CC + Enramycin at 10 ppm (positive control-PC). Challenge consisted of 15× the recommended dose of coccidiosis vaccine at placement, and Clostridium perfringens (108 CFU/bird) inoculation at 10, 11, and 12 d. Macro and microscopic evaluation, immunohistochemistry, and gene expression were evaluated at 7, 14, 21, and 28 d of age. Experiment 2. A total of 1,120 one-day old male Cobb 500 chicks were placed in floor pens with fresh litter in a completely randomized design, with 4 treatment groups, 8 replicate pens per treatment, and 35 birds per pen. The treatments were: Control, supplementation of MUR at 25,000 or 45,000 LSU(F)/kg, and a positive control (basal diet plus Enramycin). At 10, 11, and 12 d of the experiment all the birds were inoculated by oral gavage with a fresh broth culture of a field isolate Clostridium perfringens (0.5 mL containing 106 CFU/bird). It was observed that in Experiment 1 MUR supplementation reduced the infiltration of macrophages and CD8+ lymphocytes in the liver and ileum of infected birds, downregulated IL-8 and upregulated IL-10 expression. In Experiment 2, MUR linearly improved the growth performance of the birds, increased breast meat yield, and improved absorption capacity. MUR supplementation elicited an anti-inflammatory response in birds undergoing a NE challenge model that may explain the improved growth performance of supplemented birds.

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.

[Study on biofilm formation and heterogeneity in Clostridium perfringens].

Many bacteria form biofilms and survive in the actual environment. Biofilms are not only a major form of bacteria but are also involved in tolerance to environmental stresses and antibiotics, suggesting the association with bacterial pathogenesis. Cells within biofilms display phenotypic heterogeneity; thus, even bacteria, unicellular organisms, can functionally differentiate and show multicellular behavior. Therefore, it is necessary to understand bacteria as a population to control their survival and pathogenesis in the actual environment. Previously, we found that Clostridium perfringens, an anaerobic pathogenic bacterium, form different structures in different temperatures and phenotypic heterogeneity on biofilm matrix gene expression within the biofilm. In this article, I summarize the results of our research on biofilms and their heterogeneity, the mechanisms of post-transcriptional gene expression regulation of virulence genes, and bacteria-host interactions mediated by extracellular membrane vesicles.

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.

Cellular Immune Responses in Lymphoid Tissues of Broiler Chickens Experimentally Infected with Necrotic Enteritis-Producing Clostridium perfringens Strains.

Host cellular responses against Clostridium perfringens (CP), the causative agent of necrotic enteritis (NE) in chickens, are poorly understood. In the present study, we first tested the NE-producing ability of seven netB+ CP strains (CP5, CP18, CP26, CP64, CP67, CP68, and NCNE-1), using an experimental infection model of broiler chickens. Evaluation of intestinal gross lesions showed that all the strains, except CP5, were able to produce NE, while CP26 and CP64 strains produced relatively more severe lesions when compared with other groups. Next, cellular responses in the cecal tonsil (CT), bursa of Fabricius, and spleen were evaluated in chickens infected with strains representing variation in the level of virulence, namely, avirulent CP5, virulent CP18, and a relatively more virulent CP26 strain. Immunophenotyping analysis showed that CT or splenic macrophage frequencies were significantly higher in CP18- and CP26-infected chickens compared with uninfected controls, while the frequencies of γδ T-cells and B-cells in the CT of CP26-infected chickens were significantly higher than those in the uninfected, CP5- or CP18-infected groups. The T-cell analysis showed that chickens infected with CP18 and CP26 had a significantly higher number of splenic CD4+ and CD8+ T-cells expressing CD44 and CD28 activation molecules, while CP26-infected chickens also had significantly increased CT frequency of these activated CD4+ and CD8+ T-cells when compared with uninfected or CP5-infected groups. Collectively, our findings suggested that cellular responses, including activation of T-cells, are selectively induced against virulent CP strains and that the NE-producing characteristics of this pathogen may influence the outcome of immunity to NE.

Respuestas inmunes celulares en tejidos linfoides de pollos de engorde infectados experimentalmente con cepas de Clostridium perfringens productoras de enteritis necrótica. Las respuestas celulares del huésped contra Clostridium perfringens (CP), el agente causante de la enteritis necrótica (NE) en pollos, son poco conocidas. En el presente estudio, primero se analizó la capacidad de producción de enteritis necrótica de siete cepas de C. perfringens netB+ (CP5, CP18, CP26, CP64, CP67, CP68 y NCNE-1), utilizando un modelo de infección experimental de pollos de engorde. La evaluación de las lesiones macroscópicas intestinales mostró que todas las cepas, excepto CP5, podían producir enteritis necrótica, mientras que las cepas CP26 y CP64 produjeron lesiones relativamente más severas en comparación con los otros grupos. Posteriormente, se evaluaron las respuestas celulares en las tonsilas cecales (CT), la bolsa de Fabricio y en el bazo de pollos infectados con cepas que representan variaciones en el nivel de virulencia, por ejemplo las cepas CP5 avirulenta, CP18 virulenta y la cepa CP26 relativamente más virulenta. El análisis de inmunofenotipado mostró que las frecuencias de los macrófagos esplénicos o de las tonsilas cecales fueron significativamente más altas en los pollos infectados con las cepas CP18 y CP26 en comparación con los controles no infectados, mientras que las frecuencias de células T γd y células B en tonsilas cecales de los pollos infectados con la cepa CP26 fueron significativamente más altas que las de los pollos de los grupos no infectados, o infectados con las cepas CP5 o CP18. El análisis de células T mostró que los pollos infectados con las cepas CP18 y CP26 tenían un número significativamente mayor de células esplénicas T CD4+ y CD8+ que expresaban moléculas de activación CD44 y CD28, mientras que los pollos infectados con la cepa CP26 también tenían una frecuencia significativamente mayor en las tonsilas cecales de estas células T CD4+ y CD8+ activadas en comparación con grupos no infectados o infectados con la cepa CP5. En conjunto, estos hallazgos sugirieron que las respuestas celulares, incluida la activación de las células T, se inducen selectivamente contra las cepas virulentas de C. perfringens y que las características productoras de enteritis necrótica de este patógeno pueden influir en el resultado de la inmunidad contra la enteritis necrótica.

Molecular characterization and function of JAK/STAT pathway in IPEC-J2 cells during Clostridium perfringens beta2 toxin stimulation.

Intestinal infection with C. perfringens is responsible for outbreaks of diarrhea in piglets. Janus kinase / signal transducer and activator of transcription (JAK/STAT) is a vital signaling pathway that regulates cellular activity and inflammatory response, closely correlated with multiple diseases development and advances. Currently, the potential effect of JAK/STAT on C. perfringens beta2 (CPB2) treatment on porcine intestinal epithelial (IPEC-J2) cells has not been explored. The expression of JAK/STAT genes or proteins in IPEC-J2 cells induced by CPB2 were observed by qRT-PCR and Western blot, and further used WP1066 to explore the effect of JAK2/STAT3 on mechanism employed by CPB2 on apoptosis, cytotoxicity, oxidative stress and inflammatory cytokines of IPEC-J2 cells. JAK2, JAK3, STAT1, STAT3, STAT5A and STAT6 were highly expressed in CPB2-induced IPEC-J2 cells, among which STAT3 had the highest expression. Moreover, apoptosis, cytotoxicity and oxidative stress were attenuated via blocking the activation of JAK2/STAT3 by using WP1066 in CPB2-treated IPEC-J2 cells. Furthermore, WP1066 significantly suppressed the secretion of interleukin (IL)-6, IL-1ß and TNF-α induced by CPB2 in IPEC-J2 cells.Our findings provide some insights into the functional roles of JAK2/STAT3 in piglets against to C. perfringens infection.

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.

Host-mediated beneficial effects of phytochemicals for prevention of avian coccidiosis.

Both in vitro and in vivo studies were conducted to evaluate the beneficial effects of green tea extract (GT), cinnamon oil (CO), and pomegranate extract (PO) on avian coccidiosis. In experiment (EXP) 1, an in vitro culture system was used to investigate the individual effects of GT, CO, and PO on the proinflammatory cytokine response and integrity of tight junction (TJ) in chicken intestinal epithelial cells (IEC), on the differentiation of quail muscle cells and primary chicken embryonic muscle cells, and anticoccidial and antibacterial activities against Eimeria tenella sporozoites and Clostridium perfringens bacteria, respectively. In EXP 2 and 3, in vivo trials were carried out to study the dose-dependent effect of blended phytochemicals (GT, CO, PO) on coccidiosis in broiler chickens infected with E. maxima. For EXP 2, one hundred male broiler chickens (0-day-old) were allocated into the following five treatment groups: Control group for non-infected chickens (NC), Basal diet group for E. maxima-infected chickens (PC), PC group supplemented with phytochemicals at 50 (Phy 50), 100 (Phy 100), and 200 (Phy 200) mg/kg feed diets for E. maxima-infected chickens. For EXP 3, one hundred twenty male broiler chickens (0-day-old) were allocated into the following six treatment groups: NC, PC, PC supplemented with phytochemicals at 10 (Phy 10), 20 (Phy 20), 30 (Phy 30), and 100 (Phy 100) mg/kg feed for E. maxima-infected chickens. Body weights (BW) were measured on days 0, 7, 14, 20, and 22, and jejunum samples were used to measure cytokine, TJ protein, and antioxidant enzyme responses at 8 days post-infection (dpi). Fecal samples for oocyst enumeration were collected from 6 to 8 dpi. In vitro, CO and PO reduced LPS-induced IL-1ß and IL-8 in IEC, respectively, and GT enhanced the gene expression of occludin in IEC. PO at 1.0 and 5.0 mg/mL exerted antimicrobial effect against E. tenella sporozoites and C. perfringens bacteria, respectively. In vivo, chickens fed a diet supplemented with phytochemicals showed enhanced BW, reduced oocyst shedding, and decreased proinflammatory cytokines following E. maxima challenge. In conclusion, the combination of GT, CO, and PO in the diet of broiler chickens infected with E. maxima induced enhanced host disease resistance including innate immunity and gut health, which contributed to improved growth and reduced disease responses. These findings provide scientific support for the development of a novel phytogenic feed additive formula that enhances the growth and intestinal health of broiler chickens infected with coccidiosis.

Enterotoxemia produced by lambda toxin-positive Clostridium perfringens type D in 2 neonatal goat kids.

Enterotoxemia caused by Clostridium perfringens type D usually affects sheep and goats ≥ 2-wk-old. The main clinical signs and lesions of the disease are produced by the epsilon toxin (ETX) elaborated by this microorganism. However, ETX is produced in the form of a mostly inactive prototoxin that requires protease cleavage for activation. It has traditionally been believed that younger animals are not affected by type D enterotoxemia given the low trypsin activity in the intestinal content associated with the trypsin-inhibitory action of colostrum. Two Nigerian dwarf goat kids, 2- and 3-d-old, with a history of acute diarrhea followed by death, were submitted for postmortem examination and diagnostic workup. Autopsy and histopathology revealed mesocolonic edema, necrosuppurative colitis, and protein-rich pulmonary edema. Alpha toxin and ETX were detected in intestinal content, and C. perfringens type D was isolated from the colon of both animals. The isolates encoded the gene for lambda toxin, a protease that has been shown previously to activate ETX in vitro. Type D enterotoxemia has not been reported previously in neonatal kids, to our knowledge, and we suggest that lambda toxin activated the ETX.

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.

Comparative efficacy of tannin-free grain sorghum varieties for the control of necrotic enteritis caused by Clostridium perfringens in broiler chickens.

A 28-day battery cage study was conducted to test the efficacy of tannin-free grain sorghum varieties fed to Cobb 500 male broiler chickens (n = 512) and challenged with Eimeria maxima (EM) and Clostridium perfringens (CP). Birds were fed 1 of 8 treatments (corn, red/bronze, white/tan, or U.S. No. 2 sorghum) and were grouped by challenge method (challenged with EM/CP or unchallenged). On d 14, birds in the challenge group were orally inoculated with ∼5,000 oocysts of EM, and on d 19, 20, and 21, birds were given a broth culture of CP with ∼108 CFU/mL once daily. On d 21, three birds were scored for the degree/presence of necrotic enteritis (NE) lesions. Birds and feed were group weighed (d 0, 14, 21, and 28) to calculate average feed intake (FI), body weight gain (BWG), and adjusted feed conversion ratio (AdjFCR). Intestinal integrity was assessed through histological analysis of intestinal tissues, and change in transcriptome was determined using mRNA-sequencing on intestinal mucosa. Relative concentrations of secondary metabolites in grain sorghum were determined by LC-MS/MS analysis. Data were analyzed as a 2-way ANOVA with factors of treatment, challenge and their interaction. Regardless of challenge from 14 to 21 d, birds on the corn, white/tan, and U.S. No. 2 treatments were more efficient than those fed red/bronze treatment (P = 0.0026). From 14 to 28 d, BWG was significantly higher for the white/tan treatment (P = 0.024) compared to the red/bronze treatment. At 21 d, a significant interaction was observed for lesion score (P = 0.0001) in which, challenged birds fed red/bronze and white/tan treatments had reduced intestinal lesions compared to U.S. No. 2 and corn treatments. No differences among treatments were observed in jejunum morphology, but differential expression analysis showed an upregulation in defense response to bacteria and biotic stress in the challenged red/bronze treatment compared to the challenged corn. This study demonstrated improved gut health and minimal impact on growth and efficiency of broilers fed select grain sorghum varieties when challenged with EM/CP.

Comparative efficacy of postbiotic, probiotic, and antibiotic against necrotic enteritis in broiler chickens.

Prevention of necrotic enteritis (NE), caused by Clostridium perfringens (C. perfringens), is one of the most important goals to improve the profitability of broiler chickens. This work aimed to compare the efficacy of 2 antibiotic alternatives including a postbiotic (dry feed additive and aqueous nonviable Lactobacillus (L.) species fermentation) and a probiotic (dry feed additive and aqueous Bacillus (B.) subtilis and B. lischeniformis mixture) with an antibiotic (amoxicillin in water) against NE. Four hundred, day-old broiler chicks were divided into 8 equal groups (Gs), n = 50 each (5 replicates; 10 each). Chickens of G1 (postbiotic dry-feed additive), G2 (postbiotic and antibiotic in drinking water), G3 (postbiotic dry and aqueous), G4 (probiotic dry-feed additive), G5 (probiotic and antibiotic in drinking water), G6 (probiotic dry and aqueous), and G7 (nontreated) were orally inoculated with a toxigenic C. perfringens type A on the d 19 to 21 of age and predisposed with 3X coccidial vaccine for induction of NE. However, chickens of G8 were kept nontreated or challenged. The severity of NE signs was markedly decreased in G3 in comparison with other challenged treatment groups, and the mortality rates were 22%, 10%, 16%, 22%, 12%, 20%, and 36% in Gs 1, 2, 3, 4, 5, 6, and 7, respectively. The best significant (P ≤ 0.05) feed conversion ratio was detected in G3 (1.51), G6 (1.54), and G2 and G8 (1.61). In addition, the European production efficiency factor was significantly (P ≤ 0.05) improved in G3 (279.33) and G2 (266.67), but it was decreased in G7 (177.33) when compared with G8 (339.33). An improvement in intestinal and hepatic pathology and liver function tests, as well as a significant (P ≤ 0.05) decrease in bacterial counts were observed in Gs 2, 5, 3, 6, 1, and 4, respectively in comparison with G7. Immunologically, the highest significant (P ≤ 0.05) hemagglutination inhibition antibody titers for Newcastle disease virus vaccine were in Gs 1 and 3 (6.4 log2). In conclusion, the combined feed and water postbiotic treatment demonstrated promising results in ameliorating the severity of NE and improving the hepatic and the immune status of broiler chickens when compared with the commonly used probiotic and antibiotic.

Antimicrobial activity of sophorolipids against Eimeria maxima and Clostridium perfringens, and their effect on growth performance and gut health in necrotic enteritis.

The in vitro antimicrobial activity of sophorolipids (SLs) against Eimeria maxima and Clostridium perfringens, and the in vivo effects of SLs on growth performance and gut health in necrotic enteritis (NE)-afflicted broiler chickens were studied. To test the direct killing effects of SLs on enteric pathogens, 2.5 × 105 freshly prepared sporozoites of each Eimeria acervulina, E. maxima, and E. tenella were placed in each well of a 96-well plate, and the vegetative stage of Clostridium perfringens was prepared at 1 × 109 cfu/well. Four different SLs (C18:1 lactonic diacetyled SL [SL1], C18:1 deacetyled SL [SL2], C18:1 monoacetyled SL [SL3], and C18:1 diacetyled SL [SL4]), and 2 anticoccidial chemical controls, decoquinate and monensin, were evaluated at 3 dose levels (125 µg/mL, 250 µg/mL, and 500 µg/mL). Samples were incubated at 41°C for 3 h, and microbial survival ratios were measured by using a cell counter to quantify the number of live microbes stained by fluorescent dye. A total of 336 (0-day-old) male commercial broiler chickens were used to assess the effects of SLs in vivo. Chickens were randomly allocated to 6 treatment groups (7 chickens per cage, 8 cages per treatment) as follows: a control group which received a basal diet (CON), a negative control group (NC) which received a basal diet and NE challenge, and 4 SL treatment groups with NE (NC+SL1, NC+SL2, NC+SL3, and NC+SL4). The inclusion rates of SLs in each group were 200 mg/kg of feed. NE-induced chickens were orally infected with E. maxima (10,000 oocysts/chicken) on d 14, followed by C. perfringens (1 × 109 cfu/chicken) on d 19. Disease parameters measured included gut lesion scores, intestinal cytokine production, and level of tight junction protein expression. Data were analyzed using a Mixed Model (PROC MIXED) in SAS. In vitro (Experiment 1), all SLs dose-dependently decreased (P < 0.001) the viability of the three species of Eimeria sporozoites and C. perfringens. In vivo (Experiment 2), dietary SLs increased (P < 0.001) body weight and average daily gain of broiler chickens infected with NE. Dietary SL1 and SL4s increased (P < 0.05) feed conversion ratio compared to NC. Furthermore, SL1 and SL4 decreased (P < 0.05) gut lesion scores in combination with increased expression of IL1ß, IL8, TNFSF15, and IL10 genes (P < 0.05) in NE-afflicted chickens. Overall, dietary SLs promoted growth performance, intestinal immune responses, and intestinal barrier integrity of NE-afflicted, young broiler chickens.

A stress-induced model of acute necrotic enteritis in broiler chickens using dietary corticosterone administration.

Mounting evidence indicates that stress can predispose chickens to disease. The objective of the current study was to develop a method that utilized physiological stress to predispose Ross 308 broiler chickens to acute necrotic enteritis (NE). Stress was mediated through the administration of the stress hormone, corticosterone. At 11 d posthatch (p.h.), corticosterone (20 mg kg-1) administration commenced. At 12 and 13 d p.h., birds were orally inoculated with a virulent strain of Clostridium perfringens, and at 14 d p.h., birds were euthanized. Birds administered corticosterone exhibited decreased weight gain, and birds co-challenged with C. perfringens and corticosterone were affected to a higher degree. Necrotic lesions were present in birds inoculated with C. perfringens (33%), but a substantially higher prevalence of birds treated with C. perfringens and corticosterone in combination exhibited lesions (100%). Clostridium perfringens densities were correlated with necrotic lesion and histopathologic scores. Both C. perfringens and corticosterone challenge altered mRNA immune responses in the small intestine. In this regard, birds infected with the pathogen showed higher relative mRNA concentrations of toll-like receptor 2A (TLR2A), transforming growth factor beta 2 (TGFß2), and inducible nitric oxide synthase (INOS). Birds co-challenged with C. perfringens and corticosterone showed hindered TLR2A mRNA expression. A reduction in TLR2A responses mediated by corticosterone administration suggests that the glucocorticoid suppresses immune stimulation in jejunal mucosa, which may be the underlying cause for the increased prevalence and intensity of disease observed in corticosterone treated birds. Overall, the corticosterone stress model resulted in levels of NE comparable to other models of NE that currently exist without the use of a co-infection agent. This model may facilitate the exploration of mechanisms of stress-induced NE, and the development of effective alternatives to antibiotics.