Dietary Macleaya cordata extract supplementation improves the growth performance and gut health of broiler chickens with necrotic enteritis.

BACKGROUND: The poultry industry needs effective antibiotic alternatives to control outbreaks of necrotic enteritis (NE) caused by Clostridium perfringens. METHODS: The aim of this study was to investigate the effects of dietary supplementation with Macleaya cordata extract (MCE) on the immune function and gut microbiota of broilers with NE. A total of 288 1-day-old broiler chicks were randomly assigned to a 2 × 2 factorial arrangement with two concentrations of dietary MCE supplementation (0 or 350 mg/kg of diet) and two disease challenge statuses (control or NE). RESULTS: The results revealed that NE significantly increased the feed conversion rate (FCR), mortality, intestinal lesion score, the levels of IL-1ß, IL-17 and IFN-γ/IL-4 in serum and IL-17/IL-10 in the jejunal mucosa, mRNA levels of TLR2, IFN-γ and pIgR in the jejunum, and Clostridium perfringens concentrations in the cecum. NE significantly decreased the body weight (BW), body weight gain (BWG), jejunal villus height, V/C, mRNA level of AMPK-α1 in jejunum, IL-4 level in the jejunal mucosa and lactic acid bacteria abundance in the cecum. MCE significantly increased BW, BWG, jejunal villus height, V/C, mRNA levels of occludin, ZO-1 and AMPK-α1 in the jejunum, the levels of IgA and IgG in serum and IL-10 in the jejunal mucosa and mRNA levels of NF-κB, IL-10 and MHC-II in the jejunum. Additionally, MCE significantly decreased the FCR, mortality, intestinal lesion score, jejunal crypt depth, the levels of IFN-γ and IL-17 in serum and IL-17/IL-10 in the jejunal mucosa, Clostridium perfringens concentrations in the cecum, and mRNA levels of IL-17/IL-10 in the jejunum. Moreover, NE significantly increased the abundance of bacteria that are associated with inflammation, obesity and depression (Alistipes, Barnesiella, Intestinimonas, RF39 and UCG-005) and significantly decreased the abundance of short-chain fatty acid (SCFA)-producing bacteria (Anaerotruncus, Butyricicoccus and Bacteroides) in the cecum. MCE significantly increased the abundance of SCFA-producing bacteria (Streptococcus, Ruminococcus_torques_group and Lachnospiraceae_NK4A136_group) and significantly reduced the abundance of bacteria that are associated with inflammation and obesity (Alistipes, Barnesiella and UCG-010) in the cecum. In the cecum of broilers with NE, the relative abundance of Barnesiella and Alistipes was higher and that of Lachnoclostridium and Shuttleworthia was lower. Interestingly, these trends were reversed by the addition of MCE to the diet. Spearman correlation analysis showed that Barnesiella and Alistipes were associated with enhanced intestinal inflammation and inhibited growth performance, whereas Lachnoclostridium and Shuttleworthia were associated with anti-inflammatory effects. CONCLUSIONS: MCE ameliorated the loss of growth performance in broiler chickens with NE, probably by regulating the intestinal barrier, immune function, and gut microbiota.

Potential of a mixture of eugenol and garlic tincture to improve performance and intestinal health in broilers under necrotic enteritis challenge.

Plant extracts (PE) are gaining increased attention as potential alternatives to in-feed antimicrobials (AM) due to their known antimicrobial activities. This study was conducted to examine the potential of PE, a microencapsulated product composed of eugenol and garlic tincture as an alternative to AM-agent on performance and intestinal health in broilers under necrotic enteritis (NE) challenge. A total of 960 day-old mixed-sex Cobb 500 chicks were randomly distributed to 48-floor pens with 6 treatments replicated 8 times with 20 birds each. The 6 treatments were as follows: UC, unchallenged control; CC, challenged control; PE, challenged group plus PE; AM, challenged group plus AM; FAP, challenged group plus a full dose of AM with PE; HAP, challenged group plus a half dose of AM with PE in starter, grower and finisher phases. Birds in the challenged groups were inoculated with Eimeria spp. on d 9 and Clostridium perfringens on d 14. The body weight gain (BWG), feed intake (FI), feed conversion ratio (FCR), and livability of birds were compromised, and intestinal lesions and mortality were increased (P < 0.05) by NE challenge, illustrating a successful clinical NE challenge. Birds fed AM had higher BWG and FI, and lower FCR, mortality, and intestinal lesions compared to the CC group (P < 0.05). Birds fed PE had improved FCR (P < 0.05) and livability (5.8%) in an overall period compared to the CC group. On d 16, PE supplementation reduced ileal lesion scores in only male birds (P < 0.05). Birds fed PE had decreased Eimeria maxima and Eimeria acervulina oocyst counts in caecal content (P < 0.05). Birds fed PE had decreased Escherichia brunetti and total oocyst counts in caecal content, and E. acervulina oocyst counts in ileal content in only female birds (P < 0.05). On d 35, PE supplementation reduced variation of BW in both male and female birds and increased yellowness (b∗ value, 14.4%) in the thigh. These findings suggest the potential of PE supplementation in diets to improve the performance and intestinal health of birds under clinical NE as indicated by improved FCR, livability, uniformity, reduced ileal lesions, oocyst counts and increased skin yellowness. However, the protective effect of PE may not be apparent in the presence of AM in the feed.

A lytic bacteriophage isolate reduced Clostridium perfringens induced lesions in necrotic enteritis challenged broilers.

Background: Bacteriophages are viral predators of bacteria and are common in nature. Their host-specific infections against specific bacteria make them an attractive natural agent to control bacterial pathogens. Interest in the potential of bacteriophages as antibacterial agents in the production animal industries has increased. Methods: A total of 18 bacteriophages were isolated from Australian commercial poultry environments, from which three highly active phages were chosen for enrichment. Sequencing libraries were prepared using a Nextera XT kit (Illumina) and sequenced on an Illumina MiSeq instrument using 2 × 300 bp paired-end chemistry. The sequence data were then assembled and aligned with a2 bacteriophage as the reference. An animal trial was performed by oral gavaging Clostridium perfringens netB containing strain EHE-NE18 to the Ross 308 broiler chickens prior inoculation with Eimeria species. The chickens were raised following the management guide for Ross 308 from d 0 to d 21 and fed with starter and grower diets met the specific breed nutrient requirements. Body weight gain and feed intake were measured on d 9 and d 21 and FCR adjusted with mortality was calculated. Results: The isolated bacteriophages only had only 96.7% similarity to the most closely related, previously characterized, Clostridium bacteriophage indicated that they might represent a novel strain of bacteriophage. A "cocktail" containing the three bacteriophages was capable of lysing four known disease-inducing C. perfringens strains in vitro. Oral administration of the bacteriophages cocktail to broilers challenged with necrotic enteritis markedly alleviated intestinal necrotic lesions in the duodenum and jejunum on day 16 post-hatch. The phage treatment significantly reduced the lesion scores of birds challenged with NE (P < 0.01), and the lesion scores between birds treated with the bacteriophages and the unchallenged birds were not statistically different (P > 0.05). However, no effect on the growth performance was observed during the recorded period of days 9-21. Conclusion: These findings suggest that bacteriophage treatment is a promising approach to protect intestinal health from C. perfringens induced necrotic enteritis. Further research will be required on the dosing, route of administration, and large scale validation studies to further advance this approach to pathogen control.

Detection and Quantification of Clostridium perfringens and Eimeria spp. in Poultry Dust Using Real-Time PCR Under Experimental and Field Conditions.

Infection of poultry with Eimeria spp., the causative agent of coccidiosis, can predispose birds to necrotic enteritis (NE) caused by netB gene-positive strains of Clostridium perfringens. The detection of Eimeria spp., C. perfringens, and netB were examined in settled dust from broiler flocks under experimental and field conditions. Dust samples were collected from settle plates twice weekly from two experimental flocks inoculated with three species of pathogenic Eimeria in 9-day-old chicks, followed by netB gene-positive C. perfringens 5 days later to produce subclinical and clinical NE. A noninoculated flock was sampled weekly from day 0 and served as a control flock. An additional 227 dust samples from commercial broiler flocks were collected at the end-of-batch (6-7 wk of age; one scraped dust sample per flock). In the NE-subclinical and NE-clinical flocks, high levels of Eimeria spp. and C. perfringens were detected after inoculation followed by a gradual decline over time. In the control flock, C. perfringens and netB were detected at low levels. No significant effect of sampling location was evident on Eimeria spp., C. perfringens, and netB load within poultry houses. These results provide evidence that Eimeria spp., C. perfringens, and netB gene copies can be readily measured in poultry dust samples collected in settle plates and may provide an alternative sampling method for monitoring flock coccidiosis and NE status. Further studies are required to assess the utility for such a test in commercial flocks.

Artículo regular­Detección y cuantificación de Clostridium perfringens y Eimeria spp. en polvo de instalaciones avícolas mediante PCR en tiempo real bajo condiciones experimentales y de campo. La infección de aves comerciales con Eimeria spp., el agente causante de la coccidiosis, puede predisponer a las aves a enteritis necrótica (NE) causada por cepas de Clostridium perfringens positivas a la presencia del gene netB. La detección de Eimeria spp., C. perfringens y del gene netB se examinó en el polvo sedimentado de parvadas de pollos de engorde bajo condiciones experimentales y de campo. Se recolectaron muestras de polvo por sedimentación en placa dos veces por semana de dos parvadas experimentales inoculadas con tres especies de Eimeria patógena en pollitos de nueve días, seguidas de C. perfringens positiva al gene netB cinco días después para producir enteritis necrótica subclínica y clínica. Una parvada no inoculada se muestreó semanalmente desde el día cero y sirvió como parvada control. Se recolectaron 227 muestras adicionales de polvo de parvadas de pollos de engorde comerciales al final del lote (6 a 7 semanas de edad; una muestra de polvo por raspado por parvada). En las parvadas con enteritis necrótica subclínica y clínica, se detectaron niveles altos de Eimeria spp. y de C. perfringens después de la inoculación seguida de una disminución gradual con el tiempo. En la parvada control, C. perfringens y el gene netB se detectaron en niveles bajos. No fue evidente ningún efecto significativo de la ubicación del muestreo sobre la carga de Eimeria spp., C. perfringens y del gene netB dentro de las casetas. Estos resultados proporcionan evidencia de que las copias genéticas de Eimeria spp., C. perfringens y del gene y netB se pueden medir fácilmente en muestras de polvo de instalaciones avícolas recolectadas mediante sedimentación en placa y pueden proporcionar un método de muestreo alternativo para monitorear coccidiosis y el estado de enteritis necrótica en la parvada. Se requieren más estudios para evaluar la utilidad de tal prueba en parvadas comerciales.

Differential expression of intestinal genes in necrotic enteritis challenged broiler chickens with 2 different Clostridium perfringens strains.

The primary cause of necrotic enteritis (NE) disease in chickens is the NetB-positive Clostridium perfringens bacterium. Many factors are known to affect the severity of NE in the challenge models of broiler chickens, and one of these factors is the virulence of C. perfringens strain. This study was conducted to evaluate the effect of 2 pathogenic C. perfringens strains in a NE challenge model on gut health and mRNA expression of genes encoding apoptosis, tight junction, immunity, and nutrient transporters in broilers. Day-old Ross-308 male broilers (n = 468) were allocated in a 2 × 3 factorial arrangement of treatments with in-feed antibiotics (no or yes) and challenge (Non, C. perfringens strain NE18, and C. perfringens strain NE36) as the factors. The birds in the challenged groups were inoculated with Eimeria species on day 9 and with a fresh suspension of C. perfringens NE18 or NE36 on day 14 and 15. Sample collection was performed on 2 birds of each pen on day 16. Necrotic enteritis challenge, impaired feed conversion ratio during day 0 to 16 compared with the control group where the effect of the NE36 challenge was more severe than that with NE18 (P < 0.001). The mRNA expression of mucin-2, immunoglobulin-G, occludin (P < 0.001), and tight junction protein-1 (P < 0.05) genes were downregulated in both challenged groups compared with the nonchallenged counterparts. Antibiotic supplementation, on the other hand, increased weight gain, and feed intake in all challenged birds (P < 0.01), but upregulated mucin-5ac and alanine, serine, cysteine, and threonine transporter-1 (P < 0.05) only in the NE18 challenged birds. The challenge with NE36 significantly upregulated caspase-8 and claudin-1 (P < 0.001), but downregulated glucose transporter-2 (P < 0.001) compared with the NE18 challenge. These results suggest that NE challenge is detrimental to the performance of broilers through compromised intestinal health, and different C. perfringens strains can affect the severity of the disease through modulating the expression of intestinal genes encoding proteins responsible for apoptosis, gut integrity, immunity, mucus production, and nutrient transporters.

Cecal Microbiota of Free-Range Hens Varied With Different Rearing Enrichments and Ranging Patterns.

Free-range pullets are reared indoors but the adult hens can go outside which is a mismatch that may reduce adaptation in the laying environment. Rearing enrichments might enhance pullet development and adaptations to subsequent free-range housing with impact on behavior and health measures including gut microbiota. Adult free-range hens vary in range use which may also be associated with microbiota composition. A total of 1,700 Hy-Line Brown® chicks were reared indoors across 16 weeks with three enrichment treatment groups: "control" with standard litter housing, "novelty" with weekly changed novel objects, and "structural" with custom-designed perching structures in the pens. At 15 weeks, 45 pullet cecal contents were sampled before moving 1,386 pullets to the free-range housing system. At 25 weeks, range access commenced, and movements were tracked via radio-frequency identification technology. At 65 weeks, 91 hens were selected based on range use patterns ("indoor": no ranging; "high outdoor": daily ranging) across all rearing enrichment groups and cecal contents were collected for microbiota analysis via 16S rRNA amplicon sequencing at V3-V4 regions. The most common bacteria in pullets were unclassified Barnesiellaceae, Prevotella, Blautia and Clostridium and in hens Unclassified, Ruminococcus, unclassified Lachnospiraceae, unclassified Bacteroidales, unclassified Paraprevotellaceae YRC22, and Blautia. The microbial alpha diversity was not significant within the enrichment/ranging groups (pullets: P ≥ 0.17, hen rearing enrichment groups: P ≥ 0.06, hen ranging groups: P ≥ 0.54), but beta diversity significantly varied between these groups (pullets: P ≤ 0.002, hen rearing enrichment groups: P ≤ 0.001, hen ranging groups: P ≤ 0.008). Among the short-chain fatty acids (SCFAs), the propionic acid content was higher (P = 0.03) in the novelty group of pullets than the control group. There were no other significant differences in the SCFA contents between the rearing enrichment groups (all P ≥ 0.10), and the ranging groups (all P ≥ 0.17). Most of the genera identified were more abundant in the indoor than high outdoor hens. Overall, rearing enrichments affected the cecal microbiota diversity of both pullets and adult hens and was able to distinguish hens that remained inside compared with hens that ranging daily for several hours.

Effect of arabinoxylo-oligosaccharides and arabinoxylans on net energy and nutrient utilization in broilers.

Arabinoxylo-oligosaccharides (AXOS) are hydrolytic degradation products of arabinoxylans (AX) that can be fermented by the gut microbiota, thus potentially displaying prebiotic properties. This study examined the effects of AX and AXOS on net energy (NE) and nutrient utilization in broilers. Ross 308 broilers (n = 90, 30 birds per treatment) were fed wheat-soybean diets supplemented with pure AX, AXOS produced by exposing the AX to xylanase in vitro (AXOS), or AX with xylanase (AX + E) from d 10 to 21. Performance parameters were measured from d 10 to 21. On d 15, 10 birds per treatment were allocated to closed-circuit net energy chambers to assess the impact of AX and AXOS on dietary energy utilization, through assessment of both metabolisable energy (ME) and NE. Ileal and caecal digesta samples were collected on d 21 to determine the effect of AX and AXOS on ileal and total tract dry matter digestibility, ileal digestible energy, digesta pH, short chain fatty acids (SCFA) and microbiota concentration. Feed conversion ratio was numerically the lowest in birds fed the diet supplemented with AXOS, which is 1.26 compared to 1.37 and 1.30 for AX and AX + E, respectively. Ileal dry matter digestibility was higher in birds fed AXOS than those fed AX (P = 0.047). Ileal digestible energy and total tract dry matter digestibility were higher in birds fed AXOS than those fed AX or AX + E (P = 0.004 and P = 0.001, respectively). Birds fed AXOS had higher ME intake (P = 0.049) and nitrogen retention (P = 0.001) and a strong trend of higher NE (P = 0.056), NE intake (P = 0.057) and retained energy (P = 0.054) compared to those fed AX. Ileal total SCFA, lactic and formic acid concentrations were higher in birds fed AXOS than those fed AX (P = 0.011, P = 0.012 and P = 0.023, respectively). Birds fed AXOS or AX + E had higher caecal total SCFA, acetic, butyric and isovaleric acid concentrations compared to those fed AX (P = 0.001, P = 0.004, P = 0.016 and P = 0.008, respectively), and caecal propionic acid concentration was higher in birds fed AX + E than those fed AX (P = 0.050). Ileal and caecal microbiota concentrations were numerically higher and pH was lower in birds fed AXOS and AX + E than those fed AX. Results from this study indicate that feeding AXOS directly is more efficient than AXOS generation in the gastrointestinal tract, and suggest that AXOS has a potential to be an efficacious prebiotic in broiler diets.

An early feeding regime and a high-density amino acid diet on growth performance of broilers under subclinical necrotic enteritis challenge.

Broilers that have early access to feed have been shown to have enhanced immune system and gut development and heightened resilience against necrotic enteritis (NE). This study examined the effect of early feeding a high amino acid density diet on performance of broilers under a sub-clinical NE challenge model. Ross 308 broilers (n = 576) were assigned to a 2 × 2 × 2 factorial design with 2 feeding regimes (feed access either within 6 h post-hatch or after 48 h post-hatch), 2 diets (control diet or the control diet with an additional 10% digestible amino acids [HAA]) and either presence or absence of NE challenge. Oral administrations of Eimeria species (d 9) and a field strain of Clostridium perfringens (d 14) were used to induce NE. Broiler performance was analysed at d 13, 23, 30 and 35. Intestinal lesion score and bacterial count were analysed on d 16. The NE challenge reduced overall bird performance and induced severe intestinal lesions, without causing notable mortality. At d 23 bird weight was significantly lower (P < 0.001) in the challenged birds compared with the unchallenged birds, but by d 30 the challenged birds had recovered and challenge no longer had an impact on bird performance. Birds fed the HAA diet had greater body weight by d 35 and heightened Lactobacillus content in the ileum at d 16 (P < 0.05). Birds that were fed the HAA diet after a period of fasting performed better in terms of feed conversion ratio (FCR) under challenge. The findings from this study suggest there are beneficial effects of feeding high amino acid diets to birds in response to external stresses, such as post-hatch fasting and subclinical NE.