Necrotic enteritis (NE) caused by Clostridium perfringens is a reemerging disease of economic importance in areas of the world where antibiotic growth promoters have been banned. The effect of mannan-oligosaccharide (MOS) supplementation in organic diets of broilers challenged with C. perfringens on performance, gut morphology, and innate immunity was investigated. Three hundred Ross-308 broilers were fed antibiotic-free certified organic starter and grower diets. On d 14, birds were orally challenged with 1 mL of C. perfringens culture at 3 × 10(10) cfu/bird. Treatments consisted of a control no-challenge (CO; 0 g/kg of MOS in the basal diet), control challenge (COC, 0 g/kg of MOS in the basal diet), and MOS challenge (2 g/kg of MOS in the basal diet). Challenge of birds resulted in decreased feed intake and BW gain (P = 0.048 and P = 0.026, respectively). Even though supplementation of diet with MOS improved feed intake (P = 0.985), BW gain and G:F were not improved compared with those of the CO group (P = 0.026 and P = <0.001, respectively). There was no significant difference among treatments in jejunal and ileal villus height, crypt depth, and goblet cells/mm(2) (P > 0.05). Quantitative real-time PCR showed that, in the ileum, the MOS diet resulted in an upregulation of toll-like receptor (TLR)2b, TLR4, interleukin (IL)-12p35, and interferon (IFN)-γ compared with CO (P = 0.003, P = 0.018, and P = 0.024, respectively). In the cecal tonsil, challenging birds with C. perfringens resulted in an upregulation of TLR2b compared with CO (P = 0.036), and MOS resulted in an upregulation of TLR4 (P = 0.018). In conclusion, feeding a MOS-supplemented diet to C. perfringens-challenged broiler chickens did not improve performance and gut morphology-associated responses. However, MOS was capable of altering TLR and cytokine profiles, where dual TLR2 and TLR4 pathways were associated with MOS supplementation with subsequent upregulation of ileal IL-12p35 and IFN-γ, implying that MOS supplementation in C. perfringens-challenged chickens supports a proinflammatory effect via T-helper cell-1 associated pathways.

The combined effects of probiotics (Lactobacillus acidophilus, Lactobacillus casei, Streptococcus faecium, and Saccharomyces cerevisiae) and organic acids (sorbic and citric acid) on intestinal morphology and expression of immune-related genes were investigated. One-day-old chicks were randomly assigned to 1 of 3 groups: birds not receiving probiotic or organic acids (control; T1), or birds receiving an oral combination (1 g/L in water) of 10(8) CFU/g of each of the aforementioned probiotics and organic acids (1% sorbic acid and 0.2% citric acid) for 7 (T2) or 14 d (T3). Each group was divided into 5 replicate pens of 20 birds each, and 5 birds from each group (1 from each pen) were killed on d 11 and 22. Intestinal sections were collected for histological assessment, and reverse transcriptase-PCR analysis was used to assess defensin and cathelicidins expression. Quantitative real-time PCR was used to assess toll-like receptors (TLR) and cytokine expression. Duodenal villus height was greater in T2 and T3 at d 11 (P ≤ 0.036) and 22 (P ≤ 0.015) compared with T1. At d 11, duodenal goblet cell/unit area was less in T3, whereas it was greater in T2 compared with T1 in the jejunum (P = 0.009). Ileal goblet cell/unit area was greater in T3 at d 22 compared with T1 (P < 0.001). Avian beta-defensin-3 was expressed in all tissues except the bursa of T3 birds at d 11, and TLR-2 was down regulated in the cecal tonsil of birds in T2 and T3 at d 11 compared with T1 (P = 0.020 and 0.003, respectively). Expression of IL-12p35 in the ileum at d 11 was down regulated in T2 and T3 compared with T1 (P = 0.030 and 0.012, respectively). Reduced expression of INF-γ was observed in the ileum in T3 compared with T1 at d 11 (P = 0.047). Ileal IL-6 and IL-10 and cecal tonsil interferon-gamma (INF-γ) expressions were greater T2 at d 22 (P ≤ 0.047) than T1. In conclusion, supplementation of combined probiotics and organic acids resulted in inconsistent gut morphology associated responses, and avian beta-defensins and cathelicidins expression were not associated with combined probiotics and organic acids supplementation. Birds supplemented with combined probiotics and organic acids for 7 d showing similar responses in TLR-2, IL-12p35, and IFN-γ compared with those supplemented for 14 d indicates that shorter periods of supplementation might be enough to elicit beneficial responses.

The present study determined the effect of Clostridium perfringens isolates taken from necrotic enteritis (NE) outbreaks on organic farms in a NE virulence testing model. Thirteen strains were isolated in the course of the study. Six C. perfringens field isolates were taken from a naturally occurring NE outbreak on an organic farm. Polymerase chain reaction toxinotyping was used to establish C. perfringens strains, as well as to create a toxin profile. All field isolates were found to be type A and positive for alpha, beta-2 and netB toxin genes. During the NE virulence model, digesta samples were collected before oral inoculation to define the C. perfringens found as part of the natural flora. Three of the five natural flora isolates were found to be C. perfringens type E while the other two isolates were type A; only four of five isolates were positive for either netB or beta-2 toxin genes. Two isolates collected after inoculation were C. perfringens type A positive for cpb2 and netB. All isolates were tested positive for the quorum-sensing-related gene luxS, regardless of the strain source. The presence of luxS, alpha, netB and beta-2 toxin genes seems not to be a determinant of the disease as they were present in isolates from both outbreak birds as well as healthy and pre-inoculated birds. The C. perfringens field isolates induced mild NE lesions in one-half of the birds during the challenge study. Other mechanisms must play a role in the development of the disease beyond toxinotype, potentially including intestinal ecology and health, which would account for acute disease as seen in the field outbreak.