Efficacy of polymers from spontaneous carotenoid oxidation in reducing necrotic enteritis in broilers

Efficacy of polymers from spontaneous carotenoid oxidation in reducing necrotic enteritis in... ABSTRACT This study evaluated the preventive effect of the spontaneous oxidation of β-carotene (OxC-beta) in broiler chickens with necrotic enteritis by Clostridium perfringens taking into consideration various parameters including clinical signs, body weight, intestinal lesion severity, and bacterial enumeration. The mean body weight of the OxC-beta treatment groups increased significantly (P < 0.05) compared to that of the C. perfringens challenge group. Intestinal lesion scores due to C. perfringens infection were significantly alleviated by OxC-beta treatment (P < 0.05), and the number of clostridial bacteria in intestine was reduced by OxC-beta in a dose-dependent manner. OxC-beta in feed contributes to the prevention of necrotic enteritis in commercial broiler chicken, and has a positive effect in improving productivity. INTRODUCTION Beta-carotene belongs to a group of red, orange, and yellow pigments called carotenoids, which are naturally present in many fruits, vegetables, and whole grains (Mangels et al., 1993). Alpha, beta, and gamma carotene are considered provitamins as they can be converted to active vitamin A (Clemens et al., 1992). Early studies on beta-carotene and the non-vitamin A health benefits of carotenoids, especially their excellent antioxidant properties, implicated them as potential chemopreventive agents for various human chronic diseases (Peto et al., 1981). Several representative studies in humans indicated a promising role of beta-carotene in decreasing asthmatic symptoms, preventing certain cancers and heart disease, and in the treatment of AIDS, Alzheimer's disease, and rheumatoid arthritis (Peto et al., 1981; Coodley et al., 1993; Gerster, 1993; Sankaranarayanan et al., 1997; Allen et al., 2009; Li et al., 2012). Spontaneous oxidation of β-carotene (OxC-beta) produces a commercial product with a cross-section of carotenoid oxidation products containing more than 30% oxygen and 85% (w/w) beta-carotene oxygen copolymers (Johnston et al., 2014). These carotenoid-oxygen copolymers are formed by the oxidation process resulting from the addition of oxygen, and results in the formation of potentially bioactive products. Biologically active copolymers prime innate immune functions enabling a more rapid response to subsequent microbial challenges (Johnston et al., 2014). Clostridium perfringens causes necrotic enteritis (NE) in broiler chickens (Knarreborg et al., 2002). The occurrence of necrotic lesions in the small intestine is associated with proliferation of C. perfringens, which leads to more severe acute lesions. Necrotic enteritis commonly occurs in broiler chicken at around 4 weeks after hatching, and is found in many poultry production facilities worldwide (Dahiya et al., 2006; Timbermont et al., 2011). In recent years, prophylactic agents including probiotic, prebiotic, mineral, and plant extracts made of various materials have become popular on poultry farms to prevent NE. The goal of this study was to determine whether OxC-beta improves poultry productivity and prevents NE by evaluating various factors including survival rate, clinical signs, body weight, intestinal lesion severity, and bacterial enumeration. MATERIALS AND METHODS Experimental Birds We used Ross broiler chicks vaccinated with the Newcastle disease virus obtained from a commercial hatchery on day of hatch. The birds were reared in a positive pressure isolator in an environmentally controlled facility, with food and water provided ad libitum. The diet was a wheat-corn-soy-based broiler starter diet at 55%, 15%, and 22%, respectively, and further balanced following the National Research Council recommended levels for DL-methionine, L-lysine, animal-vegetable fat blend, limestone, monodicalcium phosphate, salt, trace minerals, and vitamins (NRC, 1994). Temperature of the isolator was maintained at 33–35°C for the 1st week, and subsequently at 25–28°C from week 2 to study conclusion. This study was carried out in strict compliance to legal regulations of the Laboratory Animals Act and the Animal Welfare Act of South Korea. Protocols for animal studies were approved by the Institutional Animal Care and Use Committee at Chonbuk National University. Feed additives Three feed additives were used: OxC-beta (Avivagen, Canada), bacitracin (BS Bacitrex 100, South Korea), and virginiamycin (Stafac-20, Bayer Korea, South Korea). Experimental design Birds were divided into 7 groups (n = 50 per group): 1) negative control; 2) C. perfringens challenge control; 3) OxC-beta (2 mg/kg) + C. perfringens chal-lenge; 4) OxC-beta (4 mg/kg) + C. perfringens challenge; 5) OxC-beta (6 mg/kg) + C. perfringens challenge; 6) bacitracin (55 mg/kg)+ C. perfringens challenge; and 7) virginiamycin (2 mg/kg) + C. perfringens challenge. Birds were fed the control diet or a diet that contained OxC-beta, bacitracin, or virginiamycin from day of hatch until experiment termination at 28 d. We evaluated several parameters throughout the study including performance (n = 20), bacterial culture, and development of clinical lesion (n = 10; each point). Birds were weighed individually and the mean body weight (MBW) for each group was calculated. We also determined the lesion score of each bird and calculated the mean lesion score (MLS) for each group. These experiments were replicated in parallel. Clostridium perfringens Administration Clostridium perfringens CP-013 isolated from moderate necrotic gut lesions from field cases was the challenge strain to induce signs of NE with intestinal lesions. The challenge preparation followed a previous protocol (Thompson et al., 2006). Briefly, the challenge inoculum strain was cultured in fluid thioglycolate broth (Becton Dickinson, Sparks, MD, USA), and incubated for 18 h at 37°C under anaerobic conditions. One milliliter of the fluid thioglycolate culture (1–4 × 108 CFU/mL) was orally inoculated twice daily for all bird groups (except group 1) for 3 consecutive d from days 14 to 16. Bacterial Culture To quantitatively measure populations of C. perfringens, a section of the small intestine approximately 15 cm in length, just cranial to Meckel's diverticulum, was removed. Bacterial load was determined by measuring the number of colony-forming units (CFUs) per sample. Briefly, 250 μL of intestinal digesta was loaded into the first well of each row in a 96-well plate, and 10-fold serial dilutions were made using a multichannel pipette by transferring 20 μL from the first column into 180 μL of medium in the second column, mixing 10 times, and repeating the process; pipette tips were changed between dilutions. Thereafter, 5 replicates of 10 μL from each of the 5 selected dilutions were plated onto agar using a multichannel pipette. Plates were allowed to dry and further incubated for 18 h at 37°C anaerobically. Colonies exhibiting typical morphology with more than 30 or less than 300 colonies were counted and recorded. CFUs of samples were counted individually and the average CFU for each group was calculated. Standard deviation was calculated with the mean value of each group. Performance parameters Following C. perfringens inoculation, survival rate, and clinical signs (severe depression, decreased appetite, reluctance to move, diarrhea, and ruffled feathers) were evaluated for 28 d. Body weight was evaluated at ages of 14, 21, and 28 d. Birds were weighed individually, and the mean MBW for each group was calculated. Standard deviation was calculated with the mean value of each group. Necrotic enteritis lesion score Gross lesions in the intestinal tract were graded as follows: 0, no gross lesions; 1+, thin-walled or friable small intestine; 2+, focal necrosis; 3+, larger patches of necrosis; 4+, severe, extensive necrosis typical of field cases. Lesion scores were recorded using established criteria. The lesion scores of birds were measured individually and the MLS for each group was calculated. Standard deviation was calculated with the mean value of each group. Statistical Analysis Statistical analysis was performed by ANOVA using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). P-values < 0.05 were considered statistically significant. RESULTS Growth performance Body weights were measured for birds in all 7 groups (Table 1). At 14 d, the MBW of all bird groups was not significantly different, except for birds fed 2 mg/kg virginiamycin. The MBW of birds in the OxC-beta 2, 4, and 6 mg/kg groups at 21 and 28 d was significantly increased (P < 0.05) compared to that found of the C. perfringens challenge group. At 21 and 28 d, the MBW of birds treated with antibiotics was also significantly greater (P < 0.001) compared to that of the C. perfringens challenge group. Table 1. Mean body weight of birds in each group Mean body weight (g)1 Group 1 d 14 d 21 d 28 d G1 Negative 45.0 460.2 ± 31.2b 870.2 ± 58.9b 1445.0 ± 73.8b G2 C. perfringens challenge 44.8 453.4 ± 31.1ab 810.6 ± 58.3a 1275.0 ± 84.7a G3 OxC-beta 2 mg/kg + C. perfringens challenge 44.8 459.9 ± 27.8b 855.0 ± 57.3b 1422.6 ± 85.9b G4 OxC-beta 4 mg/kg + C. perfringens challenge 44.7 460.8 ± 37.4b 845.1 ± 51.4b 1415.8 ± 83.3b G5 OxC-beta 6 mg/kg + C. perfringens challenge 44.5 441.0 ± 22.0a 846.9 ± 39.6b 1412.5 ± 98.5b G6 bacitracin 55 mg/kg + C. perfringens challenge 44.4 462.3 ± 26.0b 960.0 ± 70.1c 1627.7 ± 117.6c G7 virginiamycin 2 mg/kg + C. perfringens challenge 44.5 500.5 ± 32.4c 950.5 ± 80.0c 1616.6 ± 105.9c Mean body weight (g)1 Group 1 d 14 d 21 d 28 d G1 Negative 45.0 460.2 ± 31.2b 870.2 ± 58.9b 1445.0 ± 73.8b G2 C. perfringens challenge 44.8 453.4 ± 31.1ab 810.6 ± 58.3a 1275.0 ± 84.7a G3 OxC-beta 2 mg/kg + C. perfringens challenge 44.8 459.9 ± 27.8b 855.0 ± 57.3b 1422.6 ± 85.9b G4 OxC-beta 4 mg/kg + C. perfringens challenge 44.7 460.8 ± 37.4b 845.1 ± 51.4b 1415.8 ± 83.3b G5 OxC-beta 6 mg/kg + C. perfringens challenge 44.5 441.0 ± 22.0a 846.9 ± 39.6b 1412.5 ± 98.5b G6 bacitracin 55 mg/kg + C. perfringens challenge 44.4 462.3 ± 26.0b 960.0 ± 70.1c 1627.7 ± 117.6c G7 virginiamycin 2 mg/kg + C. perfringens challenge 44.5 500.5 ± 32.4c 950.5 ± 80.0c 1616.6 ± 105.9c 1The superscripts a, b, c denote a significant difference statistically (1-way ANOVA, SPSS 12.0). Standard deviation was calculated with the mean value of each group. View Large Table 1. Mean body weight of birds in each group Mean body weight (g)1 Group 1 d 14 d 21 d 28 d G1 Negative 45.0 460.2 ± 31.2b 870.2 ± 58.9b 1445.0 ± 73.8b G2 C. perfringens challenge 44.8 453.4 ± 31.1ab 810.6 ± 58.3a 1275.0 ± 84.7a G3 OxC-beta 2 mg/kg + C. perfringens challenge 44.8 459.9 ± 27.8b 855.0 ± 57.3b 1422.6 ± 85.9b G4 OxC-beta 4 mg/kg + C. perfringens challenge 44.7 460.8 ± 37.4b 845.1 ± 51.4b 1415.8 ± 83.3b G5 OxC-beta 6 mg/kg + C. perfringens challenge 44.5 441.0 ± 22.0a 846.9 ± 39.6b 1412.5 ± 98.5b G6 bacitracin 55 mg/kg + C. perfringens challenge 44.4 462.3 ± 26.0b 960.0 ± 70.1c 1627.7 ± 117.6c G7 virginiamycin 2 mg/kg + C. perfringens challenge 44.5 500.5 ± 32.4c 950.5 ± 80.0c 1616.6 ± 105.9c Mean body weight (g)1 Group 1 d 14 d 21 d 28 d G1 Negative 45.0 460.2 ± 31.2b 870.2 ± 58.9b 1445.0 ± 73.8b G2 C. perfringens challenge 44.8 453.4 ± 31.1ab 810.6 ± 58.3a 1275.0 ± 84.7a G3 OxC-beta 2 mg/kg + C. perfringens challenge 44.8 459.9 ± 27.8b 855.0 ± 57.3b 1422.6 ± 85.9b G4 OxC-beta 4 mg/kg + C. perfringens challenge 44.7 460.8 ± 37.4b 845.1 ± 51.4b 1415.8 ± 83.3b G5 OxC-beta 6 mg/kg + C. perfringens challenge 44.5 441.0 ± 22.0a 846.9 ± 39.6b 1412.5 ± 98.5b G6 bacitracin 55 mg/kg + C. perfringens challenge 44.4 462.3 ± 26.0b 960.0 ± 70.1c 1627.7 ± 117.6c G7 virginiamycin 2 mg/kg + C. perfringens challenge 44.5 500.5 ± 32.4c 950.5 ± 80.0c 1616.6 ± 105.9c 1The superscripts a, b, c denote a significant difference statistically (1-way ANOVA, SPSS 12.0). Standard deviation was calculated with the mean value of each group. View Large Assessment of gross lesion Following the C. perfringens challenge, intestinal lesions were scored for NE on day 21 and 28 (Table 2). We found that MLS of the negative control group was 0.30 ± 0.39, whereas that of the C. perfringens challenge group was the highest at 1.54 ± 0.58 of all bird groups. The small intestinal lesions of all bird groups showed little or multifocal hyperemia and/or hemorrhages on the mucosa of the small intestine. Intestinal lesions of all OxC-beta and antibiotics groups were significantly (P < 0.001) improved compared to the C. perfringens challenge group. This improvement of intestinal lesions correlated with a reduction for C. perfringens of the birds fed OxC-beta or antibiotics. Table 2. Intestinal lesion scores for each bird group Mean lesion score1 Group 21 d 28 d Mean G1 Negative 0.17 ± 0.41a 0.33 ± 0.48a 0.30 ± 0. 39a G2 C. perfringens challenge 1.50 ± 0.55b 1.54 ± 0.59b 1.54 ± 0.58b G3 OxC-beta 2 mg/kg + C. perfringens challenge 0.67 ± 0.52a 0.33 ± 0.48a 0.40 ± 0.51a G4 OxC-beta 4 mg/kg + C. perfringens challenge 0.67 ± 0.82a 0.50 ± 0.66a 0.53 ± 0.65a G5 OxC-beta 6 mg/kg + C. perfringens challenge 0.33 ± 0.52a 0.54 ± 0.59a 0.50 ± 0.56a G6 bacitracin 55 mg/kg + C. perfringens challenge 0.50 ± 0.52a 0.46 ± 0.59a 0.47 ± 0.54a G7 virginiamycin 2 mg/kg + C. perfringens challenge 0.83 ± 0.75ab 0.42 ± 0.50a 0.50 ± 0.57a Mean lesion score1 Group 21 d 28 d Mean G1 Negative 0.17 ± 0.41a 0.33 ± 0.48a 0.30 ± 0. 39a G2 C. perfringens challenge 1.50 ± 0.55b 1.54 ± 0.59b 1.54 ± 0.58b G3 OxC-beta 2 mg/kg + C. perfringens challenge 0.67 ± 0.52a 0.33 ± 0.48a 0.40 ± 0.51a G4 OxC-beta 4 mg/kg + C. perfringens challenge 0.67 ± 0.82a 0.50 ± 0.66a 0.53 ± 0.65a G5 OxC-beta 6 mg/kg + C. perfringens challenge 0.33 ± 0.52a 0.54 ± 0.59a 0.50 ± 0.56a G6 bacitracin 55 mg/kg + C. perfringens challenge 0.50 ± 0.52a 0.46 ± 0.59a 0.47 ± 0.54a G7 virginiamycin 2 mg/kg + C. perfringens challenge 0.83 ± 0.75ab 0.42 ± 0.50a 0.50 ± 0.57a 1The superscripts a, b, c denote a significant difference statistically (1-way ANOVA, SPSS 12.0). Standard deviation was calculated with the mean value of each group. View Large Table 2. Intestinal lesion scores for each bird group Mean lesion score1 Group 21 d 28 d Mean G1 Negative 0.17 ± 0.41a 0.33 ± 0.48a 0.30 ± 0. 39a G2 C. perfringens challenge 1.50 ± 0.55b 1.54 ± 0.59b 1.54 ± 0.58b G3 OxC-beta 2 mg/kg + C. perfringens challenge 0.67 ± 0.52a 0.33 ± 0.48a 0.40 ± 0.51a G4 OxC-beta 4 mg/kg + C. perfringens challenge 0.67 ± 0.82a 0.50 ± 0.66a 0.53 ± 0.65a G5 OxC-beta 6 mg/kg + C. perfringens challenge 0.33 ± 0.52a 0.54 ± 0.59a 0.50 ± 0.56a G6 bacitracin 55 mg/kg + C. perfringens challenge 0.50 ± 0.52a 0.46 ± 0.59a 0.47 ± 0.54a G7 virginiamycin 2 mg/kg + C. perfringens challenge 0.83 ± 0.75ab 0.42 ± 0.50a 0.50 ± 0.57a Mean lesion score1 Group 21 d 28 d Mean G1 Negative 0.17 ± 0.41a 0.33 ± 0.48a 0.30 ± 0. 39a G2 C. perfringens challenge 1.50 ± 0.55b 1.54 ± 0.59b 1.54 ± 0.58b G3 OxC-beta 2 mg/kg + C. perfringens challenge 0.67 ± 0.52a 0.33 ± 0.48a 0.40 ± 0.51a G4 OxC-beta 4 mg/kg + C. perfringens challenge 0.67 ± 0.82a 0.50 ± 0.66a 0.53 ± 0.65a G5 OxC-beta 6 mg/kg + C. perfringens challenge 0.33 ± 0.52a 0.54 ± 0.59a 0.50 ± 0.56a G6 bacitracin 55 mg/kg + C. perfringens challenge 0.50 ± 0.52a 0.46 ± 0.59a 0.47 ± 0.54a G7 virginiamycin 2 mg/kg + C. perfringens challenge 0.83 ± 0.75ab 0.42 ± 0.50a 0.50 ± 0.57a 1The superscripts a, b, c denote a significant difference statistically (1-way ANOVA, SPSS 12.0). Standard deviation was calculated with the mean value of each group. View Large Clostridium perfringens counts Bacteria in intestinal digesta were enumerated on days 10, 21, and 28 (Table 3). On day 10, the number of C. perfringens isolated from all birds prior to challenge was 102 to 103 CFU/g. On day 21, bacteria from the bird groups fed OxC-beta was significantly decreased (P < 0.05) relative to the C. perfringens challenge group. On day 28, the clostridial bacteria from the bird groups fed OxC-beta showed a greater decrease (P < 0.001) relative to the C. perfringens challenge group. In this experiment, OxC-beta and antibiotics significantly reduced the population of clostridia in the small intestine in birds from treated groups. Table 3. Mean number of colony-forming units per gram of chicken intestinal digesta for each bird group Mean CFU per gram1 of chicken intestinal digesta Group 10 d 21 d 28 d G1 Negative 4.4 × 102 ± 8.9 × 101 1.0 × 103 ± 2.7 × 102[a] 8.3 × 105 ± 2.0 × 105[a] G2 C. perfringens challenge 1.0 × 103 ± 2.7 × 102 2.0 × 105 ± 5.0 × 104[b] 1.1 × 107 ± 9.2 × 105[b] G3 OxC-beta 2 mg/kg + C. perfringens challenge 1.0 × 103 ± 2.8 × 102 7.5 × 103 ± 1.9 × 103[a] 1.7 × 105 ± 2.4 × 104[a] G4 OxC-beta 4 mg/kg + C. perfringens challenge 4.5 × 102 ± 1.3 × 102 1.2 × 103 ± 2.0 × 102[a] 3.4 × 104 ± 7.3 × 103[a] G5 OxC-beta 6 mg/kg + C. perfringens challenge 1.3 × 103 ± 2.3 × 102 2.3 × 102 ± 5.7 × 101[a] 7.1 × 104 ± 1.4 × 104[a] G6 bacitracin 55 mg/kg + C. perfringens challenge 8.6 × 102 ± 1.6 × 102 2.5 × 101 ± 9.6[c] ND G7 virginiamycin 2 mg/kg + C. perfringens challenge 1.1 × 103 ± 2.2 × 102 ND ND Mean CFU per gram1 of chicken intestinal digesta Group 10 d 21 d 28 d G1 Negative 4.4 × 102 ± 8.9 × 101 1.0 × 103 ± 2.7 × 102[a] 8.3 × 105 ± 2.0 × 105[a] G2 C. perfringens challenge 1.0 × 103 ± 2.7 × 102 2.0 × 105 ± 5.0 × 104[b] 1.1 × 107 ± 9.2 × 105[b] G3 OxC-beta 2 mg/kg + C. perfringens challenge 1.0 × 103 ± 2.8 × 102 7.5 × 103 ± 1.9 × 103[a] 1.7 × 105 ± 2.4 × 104[a] G4 OxC-beta 4 mg/kg + C. perfringens challenge 4.5 × 102 ± 1.3 × 102 1.2 × 103 ± 2.0 × 102[a] 3.4 × 104 ± 7.3 × 103[a] G5 OxC-beta 6 mg/kg + C. perfringens challenge 1.3 × 103 ± 2.3 × 102 2.3 × 102 ± 5.7 × 101[a] 7.1 × 104 ± 1.4 × 104[a] G6 bacitracin 55 mg/kg + C. perfringens challenge 8.6 × 102 ± 1.6 × 102 2.5 × 101 ± 9.6[c] ND G7 virginiamycin 2 mg/kg + C. perfringens challenge 1.1 × 103 ± 2.2 × 102 ND ND 1The superscripts a, b, c denote a statistically significant difference (1-way ANOVA, SPSS 12.0) Mean values of antibiotic-treated groups (Groups 6 and 7) were excluded from statistical analysis as the number of Clostridial bacteria was either very low or not detected. Standard deviation was calculated with the mean value of each group. ND, C. perfringens in intestinal digesta was not detected. View Large Table 3. Mean number of colony-forming units per gram of chicken intestinal digesta for each bird group Mean CFU per gram1 of chicken intestinal digesta Group 10 d 21 d 28 d G1 Negative 4.4 × 102 ± 8.9 × 101 1.0 × 103 ± 2.7 × 102[a] 8.3 × 105 ± 2.0 × 105[a] G2 C. perfringens challenge 1.0 × 103 ± 2.7 × 102 2.0 × 105 ± 5.0 × 104[b] 1.1 × 107 ± 9.2 × 105[b] G3 OxC-beta 2 mg/kg + C. perfringens challenge 1.0 × 103 ± 2.8 × 102 7.5 × 103 ± 1.9 × 103[a] 1.7 × 105 ± 2.4 × 104[a] G4 OxC-beta 4 mg/kg + C. perfringens challenge 4.5 × 102 ± 1.3 × 102 1.2 × 103 ± 2.0 × 102[a] 3.4 × 104 ± 7.3 × 103[a] G5 OxC-beta 6 mg/kg + C. perfringens challenge 1.3 × 103 ± 2.3 × 102 2.3 × 102 ± 5.7 × 101[a] 7.1 × 104 ± 1.4 × 104[a] G6 bacitracin 55 mg/kg + C. perfringens challenge 8.6 × 102 ± 1.6 × 102 2.5 × 101 ± 9.6[c] ND G7 virginiamycin 2 mg/kg + C. perfringens challenge 1.1 × 103 ± 2.2 × 102 ND ND Mean CFU per gram1 of chicken intestinal digesta Group 10 d 21 d 28 d G1 Negative 4.4 × 102 ± 8.9 × 101 1.0 × 103 ± 2.7 × 102[a] 8.3 × 105 ± 2.0 × 105[a] G2 C. perfringens challenge 1.0 × 103 ± 2.7 × 102 2.0 × 105 ± 5.0 × 104[b] 1.1 × 107 ± 9.2 × 105[b] G3 OxC-beta 2 mg/kg + C. perfringens challenge 1.0 × 103 ± 2.8 × 102 7.5 × 103 ± 1.9 × 103[a] 1.7 × 105 ± 2.4 × 104[a] G4 OxC-beta 4 mg/kg + C. perfringens challenge 4.5 × 102 ± 1.3 × 102 1.2 × 103 ± 2.0 × 102[a] 3.4 × 104 ± 7.3 × 103[a] G5 OxC-beta 6 mg/kg + C. perfringens challenge 1.3 × 103 ± 2.3 × 102 2.3 × 102 ± 5.7 × 101[a] 7.1 × 104 ± 1.4 × 104[a] G6 bacitracin 55 mg/kg + C. perfringens challenge 8.6 × 102 ± 1.6 × 102 2.5 × 101 ± 9.6[c] ND G7 virginiamycin 2 mg/kg + C. perfringens challenge 1.1 × 103 ± 2.2 × 102 ND ND 1The superscripts a, b, c denote a statistically significant difference (1-way ANOVA, SPSS 12.0) Mean values of antibiotic-treated groups (Groups 6 and 7) were excluded from statistical analysis as the number of Clostridial bacteria was either very low or not detected. Standard deviation was calculated with the mean value of each group. ND, C. perfringens in intestinal digesta was not detected. View Large DISCUSSION Virginiamycin, bacitracin, and avilamycin have been used as antibiotic growth promotors, and plant extracts (essential oils, oleoresins, and flavonoids) known to have phytogenic feed additives reduce the growth of anaerobic bacteria, including clostridia (Giannenas et al., 2003; Isabel and Santos, 2009; McReynolds et al., 2009; Mountzouris et al., 2011). These additives also improve intestinal health and inhibit the growth of harmful bacteria in the intestine. Additionally, recent reports have described antibiotics and substances comparable to those found in plant extracts have antibacterial activity, are effective in improving weight gain and gastrointestinal lesions, and cause reduction of harmful bacteria (McReynolds et al., 2009). This study evaluated the preventive effect of OxC-beta in an NE broiler chicken model. Since changes in small intestine are directly related to NE, it is important to monitor shifts in the lesion score of the treatment group compared to the positive control when evaluating the efficacy of NE prevention. The MLS of the OxC-beta treatment groups were significantly lower than that of the positive control group and were similar to those found for the negative control and antibiotic groups. Importantly, if the intestinal lesion score can be reduced, infected birds are much more likely to recover from the disease, and although these birds will weigh much less when recovering from the disease, but will be marketable. When evaluating log10 CFU values of C. perfringens, bacteria were not detected in the antibiotic group at the end of the experiment, thus confirming the strong antimicrobial effect. Values from the OxC-beta treatment groups were similar to that found in the negative control group. However, the bacterial reduction effect was larger in OxC-beta 4 and 6 mg/kg groups than in the 2 mg/kg group, and decreased more than 100 times compared to the positive control group at the end of the experiment. Although antibiotics have seen a much higher bactericidal effect on C. perfringens than OxC-beta, further investigation of secondary damage that may occur as a result of destroying the balance of normal gut flora is necessary. Benefits in poultry were more pronounced in a C. perfringens challenge model of NE, a finding consistent with the innate immune priming activity of OxC-beta reported previously (Johnston et al., 2014). Mice fed OxC-beta show increased intestinal immune receptor content and the ability to modulate host innate responsiveness to bacterial infection has been assessed by evaluating the effect of OxC-beta on the early stage of cytokine response and phagocytic activity (Burton et al., 2014; Johnston et al., 2014). Indirect support for the systemic availability of OxC-beta is provided in a preliminary study to be reported elsewhere, with prima facie evidence of OxC-beta activity in vivo in exploratory animal feeding trials and OxC-beta in feed-improved measures of production efficiency (i.e., feed conversion and growth), in both swine and broiler chickens (Johnston et al., 2014). Further support for the systemic availability of orally administered OxC-beta was provided in a study showing enhanced resolution of inflammation in a model of bovine respiratory disease (Duquette et al., 2014). In this study, there was no growth promotion by OxC-beta. At 14 d, the MBW of the virginiamycin-treated group was significantly higher than that of the other groups. This is presumably due to the growth promotion effect of antibiotics; the bacitracin group showed the highest MBW after the challenge, and may be because of the combined action of growth promotion and antimicrobial effects. OxC-beta may not be as promising as antibiotics in terms of growth promotion, although weight gain may vary depending on animal age, period, and dose of substance administration. Nevertheless, the positive control group showed significant weight loss post-challenge, while all groups treated with OxC-beta maintained body weight similar to that of the negative control group. Thus, productivity improvement is expected by minimizing damage in case of NE outbreak. In summary, dietary OxC-beta supplements exerted notable effects on NE lesions and decreased C. perfringens in small intestine. Administration of 2 to 6 mg/kg in-feed OxC-beta can contribute to the prevention of NE at commercial broiler chicken farms, and may also have a positive effect in improving productivity in outbreaks of NE. ACKNOWLEDGMENTS This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (311007-5, 716002-7), South Korea and Avivagen Inc, Canada. REFERENCES Allen S. , Britton J. , Leonardi-Bee J. . 2009 . Association between antioxidant vitamins and asthma outcome measures: systematic review and meta-analysis . Thorax . 64 : 610 – 619 . Google Scholar CrossRef Search ADS PubMed Burton G. W. , Daroszewski J. , Nickerson J. G. , Johnston J. B. , Mogg T. J. , Nikiforov G. B. . 2014 . β-Carotene autoxidation: oxygen copolymerization, non-vitamin A products, and immunological activity . Can. J. Chem. 92 : 305 – 316 . Google Scholar CrossRef Search ADS Clemens M. , Müller-Ladner C. , Gey K. . 1992 . Vitamins during high dose chemo-and radiotherapy . Z. Ernährungswiss . 31 : 110 – 120 . Google Scholar CrossRef Search ADS PubMed Coodley G. O. , Nelson H. D. , Loveless M. O. , Folk C. . 1993 . [beta]-Carotene in HIV Infection . 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Assessment of a phytogenic feed additive effect on broiler growth performance, nutrient digestibility and caecal microflora composition . Anim. Feed Sci. Technol. 168 : 223 – 231 . Google Scholar CrossRef Search ADS NRC . 1994 . Nutrient Requirements of Poultry . 8th rev. ed . National Academy Press , Washington, DC . Peto R. , Doll R. , Buckley J. D. , Sporn M. . 1981 . Can dietary beta-carotene materially reduce human cancer rates? Nature . 290 : 201 – 208 . Google Scholar CrossRef Search ADS PubMed Sankaranarayanan R. , Mathew B. , Varghese C. , Sudhakaran P. , Menon V. , Jayadeep A. , Nair M. , Mathews C. , Mahalingam T. , Balaram P. . 1997 . Chemoprevention of oral leukoplakia with vitamin A and beta carotene: an assessment . Oral Oncol. 33 : 231 – 236 . Google Scholar CrossRef Search ADS PubMed Thompson D. , Parreira V. , Kulkarni R. , Prescott J. . 2006 . Live attenuated vaccine-based control of necrotic enteritis of broiler chickens . Vet. Microbiol. 113 : 25 – 34 . Google Scholar CrossRef Search ADS PubMed Timbermont L. , Haesebrouck F. , Ducatelle R. , Van Immerseel F. . 2011 . Necrotic enteritis in broilers: an updated review on the pathogenesis . Avian Pathol. 40 : 341 – 347 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Efficacy of polymers from spontaneous carotenoid oxidation in reducing necrotic enteritis in broilers

Poultry Science , Volume 97 (9) – Sep 1, 2018

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0032-5791
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Abstract

ABSTRACT This study evaluated the preventive effect of the spontaneous oxidation of β-carotene (OxC-beta) in broiler chickens with necrotic enteritis by Clostridium perfringens taking into consideration various parameters including clinical signs, body weight, intestinal lesion severity, and bacterial enumeration. The mean body weight of the OxC-beta treatment groups increased significantly (P < 0.05) compared to that of the C. perfringens challenge group. Intestinal lesion scores due to C. perfringens infection were significantly alleviated by OxC-beta treatment (P < 0.05), and the number of clostridial bacteria in intestine was reduced by OxC-beta in a dose-dependent manner. OxC-beta in feed contributes to the prevention of necrotic enteritis in commercial broiler chicken, and has a positive effect in improving productivity. INTRODUCTION Beta-carotene belongs to a group of red, orange, and yellow pigments called carotenoids, which are naturally present in many fruits, vegetables, and whole grains (Mangels et al., 1993). Alpha, beta, and gamma carotene are considered provitamins as they can be converted to active vitamin A (Clemens et al., 1992). Early studies on beta-carotene and the non-vitamin A health benefits of carotenoids, especially their excellent antioxidant properties, implicated them as potential chemopreventive agents for various human chronic diseases (Peto et al., 1981). Several representative studies in humans indicated a promising role of beta-carotene in decreasing asthmatic symptoms, preventing certain cancers and heart disease, and in the treatment of AIDS, Alzheimer's disease, and rheumatoid arthritis (Peto et al., 1981; Coodley et al., 1993; Gerster, 1993; Sankaranarayanan et al., 1997; Allen et al., 2009; Li et al., 2012). Spontaneous oxidation of β-carotene (OxC-beta) produces a commercial product with a cross-section of carotenoid oxidation products containing more than 30% oxygen and 85% (w/w) beta-carotene oxygen copolymers (Johnston et al., 2014). These carotenoid-oxygen copolymers are formed by the oxidation process resulting from the addition of oxygen, and results in the formation of potentially bioactive products. Biologically active copolymers prime innate immune functions enabling a more rapid response to subsequent microbial challenges (Johnston et al., 2014). Clostridium perfringens causes necrotic enteritis (NE) in broiler chickens (Knarreborg et al., 2002). The occurrence of necrotic lesions in the small intestine is associated with proliferation of C. perfringens, which leads to more severe acute lesions. Necrotic enteritis commonly occurs in broiler chicken at around 4 weeks after hatching, and is found in many poultry production facilities worldwide (Dahiya et al., 2006; Timbermont et al., 2011). In recent years, prophylactic agents including probiotic, prebiotic, mineral, and plant extracts made of various materials have become popular on poultry farms to prevent NE. The goal of this study was to determine whether OxC-beta improves poultry productivity and prevents NE by evaluating various factors including survival rate, clinical signs, body weight, intestinal lesion severity, and bacterial enumeration. MATERIALS AND METHODS Experimental Birds We used Ross broiler chicks vaccinated with the Newcastle disease virus obtained from a commercial hatchery on day of hatch. The birds were reared in a positive pressure isolator in an environmentally controlled facility, with food and water provided ad libitum. The diet was a wheat-corn-soy-based broiler starter diet at 55%, 15%, and 22%, respectively, and further balanced following the National Research Council recommended levels for DL-methionine, L-lysine, animal-vegetable fat blend, limestone, monodicalcium phosphate, salt, trace minerals, and vitamins (NRC, 1994). Temperature of the isolator was maintained at 33–35°C for the 1st week, and subsequently at 25–28°C from week 2 to study conclusion. This study was carried out in strict compliance to legal regulations of the Laboratory Animals Act and the Animal Welfare Act of South Korea. Protocols for animal studies were approved by the Institutional Animal Care and Use Committee at Chonbuk National University. Feed additives Three feed additives were used: OxC-beta (Avivagen, Canada), bacitracin (BS Bacitrex 100, South Korea), and virginiamycin (Stafac-20, Bayer Korea, South Korea). Experimental design Birds were divided into 7 groups (n = 50 per group): 1) negative control; 2) C. perfringens challenge control; 3) OxC-beta (2 mg/kg) + C. perfringens chal-lenge; 4) OxC-beta (4 mg/kg) + C. perfringens challenge; 5) OxC-beta (6 mg/kg) + C. perfringens challenge; 6) bacitracin (55 mg/kg)+ C. perfringens challenge; and 7) virginiamycin (2 mg/kg) + C. perfringens challenge. Birds were fed the control diet or a diet that contained OxC-beta, bacitracin, or virginiamycin from day of hatch until experiment termination at 28 d. We evaluated several parameters throughout the study including performance (n = 20), bacterial culture, and development of clinical lesion (n = 10; each point). Birds were weighed individually and the mean body weight (MBW) for each group was calculated. We also determined the lesion score of each bird and calculated the mean lesion score (MLS) for each group. These experiments were replicated in parallel. Clostridium perfringens Administration Clostridium perfringens CP-013 isolated from moderate necrotic gut lesions from field cases was the challenge strain to induce signs of NE with intestinal lesions. The challenge preparation followed a previous protocol (Thompson et al., 2006). Briefly, the challenge inoculum strain was cultured in fluid thioglycolate broth (Becton Dickinson, Sparks, MD, USA), and incubated for 18 h at 37°C under anaerobic conditions. One milliliter of the fluid thioglycolate culture (1–4 × 108 CFU/mL) was orally inoculated twice daily for all bird groups (except group 1) for 3 consecutive d from days 14 to 16. Bacterial Culture To quantitatively measure populations of C. perfringens, a section of the small intestine approximately 15 cm in length, just cranial to Meckel's diverticulum, was removed. Bacterial load was determined by measuring the number of colony-forming units (CFUs) per sample. Briefly, 250 μL of intestinal digesta was loaded into the first well of each row in a 96-well plate, and 10-fold serial dilutions were made using a multichannel pipette by transferring 20 μL from the first column into 180 μL of medium in the second column, mixing 10 times, and repeating the process; pipette tips were changed between dilutions. Thereafter, 5 replicates of 10 μL from each of the 5 selected dilutions were plated onto agar using a multichannel pipette. Plates were allowed to dry and further incubated for 18 h at 37°C anaerobically. Colonies exhibiting typical morphology with more than 30 or less than 300 colonies were counted and recorded. CFUs of samples were counted individually and the average CFU for each group was calculated. Standard deviation was calculated with the mean value of each group. Performance parameters Following C. perfringens inoculation, survival rate, and clinical signs (severe depression, decreased appetite, reluctance to move, diarrhea, and ruffled feathers) were evaluated for 28 d. Body weight was evaluated at ages of 14, 21, and 28 d. Birds were weighed individually, and the mean MBW for each group was calculated. Standard deviation was calculated with the mean value of each group. Necrotic enteritis lesion score Gross lesions in the intestinal tract were graded as follows: 0, no gross lesions; 1+, thin-walled or friable small intestine; 2+, focal necrosis; 3+, larger patches of necrosis; 4+, severe, extensive necrosis typical of field cases. Lesion scores were recorded using established criteria. The lesion scores of birds were measured individually and the MLS for each group was calculated. Standard deviation was calculated with the mean value of each group. Statistical Analysis Statistical analysis was performed by ANOVA using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). P-values < 0.05 were considered statistically significant. RESULTS Growth performance Body weights were measured for birds in all 7 groups (Table 1). At 14 d, the MBW of all bird groups was not significantly different, except for birds fed 2 mg/kg virginiamycin. The MBW of birds in the OxC-beta 2, 4, and 6 mg/kg groups at 21 and 28 d was significantly increased (P < 0.05) compared to that found of the C. perfringens challenge group. At 21 and 28 d, the MBW of birds treated with antibiotics was also significantly greater (P < 0.001) compared to that of the C. perfringens challenge group. Table 1. Mean body weight of birds in each group Mean body weight (g)1 Group 1 d 14 d 21 d 28 d G1 Negative 45.0 460.2 ± 31.2b 870.2 ± 58.9b 1445.0 ± 73.8b G2 C. perfringens challenge 44.8 453.4 ± 31.1ab 810.6 ± 58.3a 1275.0 ± 84.7a G3 OxC-beta 2 mg/kg + C. perfringens challenge 44.8 459.9 ± 27.8b 855.0 ± 57.3b 1422.6 ± 85.9b G4 OxC-beta 4 mg/kg + C. perfringens challenge 44.7 460.8 ± 37.4b 845.1 ± 51.4b 1415.8 ± 83.3b G5 OxC-beta 6 mg/kg + C. perfringens challenge 44.5 441.0 ± 22.0a 846.9 ± 39.6b 1412.5 ± 98.5b G6 bacitracin 55 mg/kg + C. perfringens challenge 44.4 462.3 ± 26.0b 960.0 ± 70.1c 1627.7 ± 117.6c G7 virginiamycin 2 mg/kg + C. perfringens challenge 44.5 500.5 ± 32.4c 950.5 ± 80.0c 1616.6 ± 105.9c Mean body weight (g)1 Group 1 d 14 d 21 d 28 d G1 Negative 45.0 460.2 ± 31.2b 870.2 ± 58.9b 1445.0 ± 73.8b G2 C. perfringens challenge 44.8 453.4 ± 31.1ab 810.6 ± 58.3a 1275.0 ± 84.7a G3 OxC-beta 2 mg/kg + C. perfringens challenge 44.8 459.9 ± 27.8b 855.0 ± 57.3b 1422.6 ± 85.9b G4 OxC-beta 4 mg/kg + C. perfringens challenge 44.7 460.8 ± 37.4b 845.1 ± 51.4b 1415.8 ± 83.3b G5 OxC-beta 6 mg/kg + C. perfringens challenge 44.5 441.0 ± 22.0a 846.9 ± 39.6b 1412.5 ± 98.5b G6 bacitracin 55 mg/kg + C. perfringens challenge 44.4 462.3 ± 26.0b 960.0 ± 70.1c 1627.7 ± 117.6c G7 virginiamycin 2 mg/kg + C. perfringens challenge 44.5 500.5 ± 32.4c 950.5 ± 80.0c 1616.6 ± 105.9c 1The superscripts a, b, c denote a significant difference statistically (1-way ANOVA, SPSS 12.0). Standard deviation was calculated with the mean value of each group. View Large Table 1. Mean body weight of birds in each group Mean body weight (g)1 Group 1 d 14 d 21 d 28 d G1 Negative 45.0 460.2 ± 31.2b 870.2 ± 58.9b 1445.0 ± 73.8b G2 C. perfringens challenge 44.8 453.4 ± 31.1ab 810.6 ± 58.3a 1275.0 ± 84.7a G3 OxC-beta 2 mg/kg + C. perfringens challenge 44.8 459.9 ± 27.8b 855.0 ± 57.3b 1422.6 ± 85.9b G4 OxC-beta 4 mg/kg + C. perfringens challenge 44.7 460.8 ± 37.4b 845.1 ± 51.4b 1415.8 ± 83.3b G5 OxC-beta 6 mg/kg + C. perfringens challenge 44.5 441.0 ± 22.0a 846.9 ± 39.6b 1412.5 ± 98.5b G6 bacitracin 55 mg/kg + C. perfringens challenge 44.4 462.3 ± 26.0b 960.0 ± 70.1c 1627.7 ± 117.6c G7 virginiamycin 2 mg/kg + C. perfringens challenge 44.5 500.5 ± 32.4c 950.5 ± 80.0c 1616.6 ± 105.9c Mean body weight (g)1 Group 1 d 14 d 21 d 28 d G1 Negative 45.0 460.2 ± 31.2b 870.2 ± 58.9b 1445.0 ± 73.8b G2 C. perfringens challenge 44.8 453.4 ± 31.1ab 810.6 ± 58.3a 1275.0 ± 84.7a G3 OxC-beta 2 mg/kg + C. perfringens challenge 44.8 459.9 ± 27.8b 855.0 ± 57.3b 1422.6 ± 85.9b G4 OxC-beta 4 mg/kg + C. perfringens challenge 44.7 460.8 ± 37.4b 845.1 ± 51.4b 1415.8 ± 83.3b G5 OxC-beta 6 mg/kg + C. perfringens challenge 44.5 441.0 ± 22.0a 846.9 ± 39.6b 1412.5 ± 98.5b G6 bacitracin 55 mg/kg + C. perfringens challenge 44.4 462.3 ± 26.0b 960.0 ± 70.1c 1627.7 ± 117.6c G7 virginiamycin 2 mg/kg + C. perfringens challenge 44.5 500.5 ± 32.4c 950.5 ± 80.0c 1616.6 ± 105.9c 1The superscripts a, b, c denote a significant difference statistically (1-way ANOVA, SPSS 12.0). Standard deviation was calculated with the mean value of each group. View Large Assessment of gross lesion Following the C. perfringens challenge, intestinal lesions were scored for NE on day 21 and 28 (Table 2). We found that MLS of the negative control group was 0.30 ± 0.39, whereas that of the C. perfringens challenge group was the highest at 1.54 ± 0.58 of all bird groups. The small intestinal lesions of all bird groups showed little or multifocal hyperemia and/or hemorrhages on the mucosa of the small intestine. Intestinal lesions of all OxC-beta and antibiotics groups were significantly (P < 0.001) improved compared to the C. perfringens challenge group. This improvement of intestinal lesions correlated with a reduction for C. perfringens of the birds fed OxC-beta or antibiotics. Table 2. Intestinal lesion scores for each bird group Mean lesion score1 Group 21 d 28 d Mean G1 Negative 0.17 ± 0.41a 0.33 ± 0.48a 0.30 ± 0. 39a G2 C. perfringens challenge 1.50 ± 0.55b 1.54 ± 0.59b 1.54 ± 0.58b G3 OxC-beta 2 mg/kg + C. perfringens challenge 0.67 ± 0.52a 0.33 ± 0.48a 0.40 ± 0.51a G4 OxC-beta 4 mg/kg + C. perfringens challenge 0.67 ± 0.82a 0.50 ± 0.66a 0.53 ± 0.65a G5 OxC-beta 6 mg/kg + C. perfringens challenge 0.33 ± 0.52a 0.54 ± 0.59a 0.50 ± 0.56a G6 bacitracin 55 mg/kg + C. perfringens challenge 0.50 ± 0.52a 0.46 ± 0.59a 0.47 ± 0.54a G7 virginiamycin 2 mg/kg + C. perfringens challenge 0.83 ± 0.75ab 0.42 ± 0.50a 0.50 ± 0.57a Mean lesion score1 Group 21 d 28 d Mean G1 Negative 0.17 ± 0.41a 0.33 ± 0.48a 0.30 ± 0. 39a G2 C. perfringens challenge 1.50 ± 0.55b 1.54 ± 0.59b 1.54 ± 0.58b G3 OxC-beta 2 mg/kg + C. perfringens challenge 0.67 ± 0.52a 0.33 ± 0.48a 0.40 ± 0.51a G4 OxC-beta 4 mg/kg + C. perfringens challenge 0.67 ± 0.82a 0.50 ± 0.66a 0.53 ± 0.65a G5 OxC-beta 6 mg/kg + C. perfringens challenge 0.33 ± 0.52a 0.54 ± 0.59a 0.50 ± 0.56a G6 bacitracin 55 mg/kg + C. perfringens challenge 0.50 ± 0.52a 0.46 ± 0.59a 0.47 ± 0.54a G7 virginiamycin 2 mg/kg + C. perfringens challenge 0.83 ± 0.75ab 0.42 ± 0.50a 0.50 ± 0.57a 1The superscripts a, b, c denote a significant difference statistically (1-way ANOVA, SPSS 12.0). Standard deviation was calculated with the mean value of each group. View Large Table 2. Intestinal lesion scores for each bird group Mean lesion score1 Group 21 d 28 d Mean G1 Negative 0.17 ± 0.41a 0.33 ± 0.48a 0.30 ± 0. 39a G2 C. perfringens challenge 1.50 ± 0.55b 1.54 ± 0.59b 1.54 ± 0.58b G3 OxC-beta 2 mg/kg + C. perfringens challenge 0.67 ± 0.52a 0.33 ± 0.48a 0.40 ± 0.51a G4 OxC-beta 4 mg/kg + C. perfringens challenge 0.67 ± 0.82a 0.50 ± 0.66a 0.53 ± 0.65a G5 OxC-beta 6 mg/kg + C. perfringens challenge 0.33 ± 0.52a 0.54 ± 0.59a 0.50 ± 0.56a G6 bacitracin 55 mg/kg + C. perfringens challenge 0.50 ± 0.52a 0.46 ± 0.59a 0.47 ± 0.54a G7 virginiamycin 2 mg/kg + C. perfringens challenge 0.83 ± 0.75ab 0.42 ± 0.50a 0.50 ± 0.57a Mean lesion score1 Group 21 d 28 d Mean G1 Negative 0.17 ± 0.41a 0.33 ± 0.48a 0.30 ± 0. 39a G2 C. perfringens challenge 1.50 ± 0.55b 1.54 ± 0.59b 1.54 ± 0.58b G3 OxC-beta 2 mg/kg + C. perfringens challenge 0.67 ± 0.52a 0.33 ± 0.48a 0.40 ± 0.51a G4 OxC-beta 4 mg/kg + C. perfringens challenge 0.67 ± 0.82a 0.50 ± 0.66a 0.53 ± 0.65a G5 OxC-beta 6 mg/kg + C. perfringens challenge 0.33 ± 0.52a 0.54 ± 0.59a 0.50 ± 0.56a G6 bacitracin 55 mg/kg + C. perfringens challenge 0.50 ± 0.52a 0.46 ± 0.59a 0.47 ± 0.54a G7 virginiamycin 2 mg/kg + C. perfringens challenge 0.83 ± 0.75ab 0.42 ± 0.50a 0.50 ± 0.57a 1The superscripts a, b, c denote a significant difference statistically (1-way ANOVA, SPSS 12.0). Standard deviation was calculated with the mean value of each group. View Large Clostridium perfringens counts Bacteria in intestinal digesta were enumerated on days 10, 21, and 28 (Table 3). On day 10, the number of C. perfringens isolated from all birds prior to challenge was 102 to 103 CFU/g. On day 21, bacteria from the bird groups fed OxC-beta was significantly decreased (P < 0.05) relative to the C. perfringens challenge group. On day 28, the clostridial bacteria from the bird groups fed OxC-beta showed a greater decrease (P < 0.001) relative to the C. perfringens challenge group. In this experiment, OxC-beta and antibiotics significantly reduced the population of clostridia in the small intestine in birds from treated groups. Table 3. Mean number of colony-forming units per gram of chicken intestinal digesta for each bird group Mean CFU per gram1 of chicken intestinal digesta Group 10 d 21 d 28 d G1 Negative 4.4 × 102 ± 8.9 × 101 1.0 × 103 ± 2.7 × 102[a] 8.3 × 105 ± 2.0 × 105[a] G2 C. perfringens challenge 1.0 × 103 ± 2.7 × 102 2.0 × 105 ± 5.0 × 104[b] 1.1 × 107 ± 9.2 × 105[b] G3 OxC-beta 2 mg/kg + C. perfringens challenge 1.0 × 103 ± 2.8 × 102 7.5 × 103 ± 1.9 × 103[a] 1.7 × 105 ± 2.4 × 104[a] G4 OxC-beta 4 mg/kg + C. perfringens challenge 4.5 × 102 ± 1.3 × 102 1.2 × 103 ± 2.0 × 102[a] 3.4 × 104 ± 7.3 × 103[a] G5 OxC-beta 6 mg/kg + C. perfringens challenge 1.3 × 103 ± 2.3 × 102 2.3 × 102 ± 5.7 × 101[a] 7.1 × 104 ± 1.4 × 104[a] G6 bacitracin 55 mg/kg + C. perfringens challenge 8.6 × 102 ± 1.6 × 102 2.5 × 101 ± 9.6[c] ND G7 virginiamycin 2 mg/kg + C. perfringens challenge 1.1 × 103 ± 2.2 × 102 ND ND Mean CFU per gram1 of chicken intestinal digesta Group 10 d 21 d 28 d G1 Negative 4.4 × 102 ± 8.9 × 101 1.0 × 103 ± 2.7 × 102[a] 8.3 × 105 ± 2.0 × 105[a] G2 C. perfringens challenge 1.0 × 103 ± 2.7 × 102 2.0 × 105 ± 5.0 × 104[b] 1.1 × 107 ± 9.2 × 105[b] G3 OxC-beta 2 mg/kg + C. perfringens challenge 1.0 × 103 ± 2.8 × 102 7.5 × 103 ± 1.9 × 103[a] 1.7 × 105 ± 2.4 × 104[a] G4 OxC-beta 4 mg/kg + C. perfringens challenge 4.5 × 102 ± 1.3 × 102 1.2 × 103 ± 2.0 × 102[a] 3.4 × 104 ± 7.3 × 103[a] G5 OxC-beta 6 mg/kg + C. perfringens challenge 1.3 × 103 ± 2.3 × 102 2.3 × 102 ± 5.7 × 101[a] 7.1 × 104 ± 1.4 × 104[a] G6 bacitracin 55 mg/kg + C. perfringens challenge 8.6 × 102 ± 1.6 × 102 2.5 × 101 ± 9.6[c] ND G7 virginiamycin 2 mg/kg + C. perfringens challenge 1.1 × 103 ± 2.2 × 102 ND ND 1The superscripts a, b, c denote a statistically significant difference (1-way ANOVA, SPSS 12.0) Mean values of antibiotic-treated groups (Groups 6 and 7) were excluded from statistical analysis as the number of Clostridial bacteria was either very low or not detected. Standard deviation was calculated with the mean value of each group. ND, C. perfringens in intestinal digesta was not detected. View Large Table 3. Mean number of colony-forming units per gram of chicken intestinal digesta for each bird group Mean CFU per gram1 of chicken intestinal digesta Group 10 d 21 d 28 d G1 Negative 4.4 × 102 ± 8.9 × 101 1.0 × 103 ± 2.7 × 102[a] 8.3 × 105 ± 2.0 × 105[a] G2 C. perfringens challenge 1.0 × 103 ± 2.7 × 102 2.0 × 105 ± 5.0 × 104[b] 1.1 × 107 ± 9.2 × 105[b] G3 OxC-beta 2 mg/kg + C. perfringens challenge 1.0 × 103 ± 2.8 × 102 7.5 × 103 ± 1.9 × 103[a] 1.7 × 105 ± 2.4 × 104[a] G4 OxC-beta 4 mg/kg + C. perfringens challenge 4.5 × 102 ± 1.3 × 102 1.2 × 103 ± 2.0 × 102[a] 3.4 × 104 ± 7.3 × 103[a] G5 OxC-beta 6 mg/kg + C. perfringens challenge 1.3 × 103 ± 2.3 × 102 2.3 × 102 ± 5.7 × 101[a] 7.1 × 104 ± 1.4 × 104[a] G6 bacitracin 55 mg/kg + C. perfringens challenge 8.6 × 102 ± 1.6 × 102 2.5 × 101 ± 9.6[c] ND G7 virginiamycin 2 mg/kg + C. perfringens challenge 1.1 × 103 ± 2.2 × 102 ND ND Mean CFU per gram1 of chicken intestinal digesta Group 10 d 21 d 28 d G1 Negative 4.4 × 102 ± 8.9 × 101 1.0 × 103 ± 2.7 × 102[a] 8.3 × 105 ± 2.0 × 105[a] G2 C. perfringens challenge 1.0 × 103 ± 2.7 × 102 2.0 × 105 ± 5.0 × 104[b] 1.1 × 107 ± 9.2 × 105[b] G3 OxC-beta 2 mg/kg + C. perfringens challenge 1.0 × 103 ± 2.8 × 102 7.5 × 103 ± 1.9 × 103[a] 1.7 × 105 ± 2.4 × 104[a] G4 OxC-beta 4 mg/kg + C. perfringens challenge 4.5 × 102 ± 1.3 × 102 1.2 × 103 ± 2.0 × 102[a] 3.4 × 104 ± 7.3 × 103[a] G5 OxC-beta 6 mg/kg + C. perfringens challenge 1.3 × 103 ± 2.3 × 102 2.3 × 102 ± 5.7 × 101[a] 7.1 × 104 ± 1.4 × 104[a] G6 bacitracin 55 mg/kg + C. perfringens challenge 8.6 × 102 ± 1.6 × 102 2.5 × 101 ± 9.6[c] ND G7 virginiamycin 2 mg/kg + C. perfringens challenge 1.1 × 103 ± 2.2 × 102 ND ND 1The superscripts a, b, c denote a statistically significant difference (1-way ANOVA, SPSS 12.0) Mean values of antibiotic-treated groups (Groups 6 and 7) were excluded from statistical analysis as the number of Clostridial bacteria was either very low or not detected. Standard deviation was calculated with the mean value of each group. ND, C. perfringens in intestinal digesta was not detected. View Large DISCUSSION Virginiamycin, bacitracin, and avilamycin have been used as antibiotic growth promotors, and plant extracts (essential oils, oleoresins, and flavonoids) known to have phytogenic feed additives reduce the growth of anaerobic bacteria, including clostridia (Giannenas et al., 2003; Isabel and Santos, 2009; McReynolds et al., 2009; Mountzouris et al., 2011). These additives also improve intestinal health and inhibit the growth of harmful bacteria in the intestine. Additionally, recent reports have described antibiotics and substances comparable to those found in plant extracts have antibacterial activity, are effective in improving weight gain and gastrointestinal lesions, and cause reduction of harmful bacteria (McReynolds et al., 2009). This study evaluated the preventive effect of OxC-beta in an NE broiler chicken model. Since changes in small intestine are directly related to NE, it is important to monitor shifts in the lesion score of the treatment group compared to the positive control when evaluating the efficacy of NE prevention. The MLS of the OxC-beta treatment groups were significantly lower than that of the positive control group and were similar to those found for the negative control and antibiotic groups. Importantly, if the intestinal lesion score can be reduced, infected birds are much more likely to recover from the disease, and although these birds will weigh much less when recovering from the disease, but will be marketable. When evaluating log10 CFU values of C. perfringens, bacteria were not detected in the antibiotic group at the end of the experiment, thus confirming the strong antimicrobial effect. Values from the OxC-beta treatment groups were similar to that found in the negative control group. However, the bacterial reduction effect was larger in OxC-beta 4 and 6 mg/kg groups than in the 2 mg/kg group, and decreased more than 100 times compared to the positive control group at the end of the experiment. Although antibiotics have seen a much higher bactericidal effect on C. perfringens than OxC-beta, further investigation of secondary damage that may occur as a result of destroying the balance of normal gut flora is necessary. Benefits in poultry were more pronounced in a C. perfringens challenge model of NE, a finding consistent with the innate immune priming activity of OxC-beta reported previously (Johnston et al., 2014). Mice fed OxC-beta show increased intestinal immune receptor content and the ability to modulate host innate responsiveness to bacterial infection has been assessed by evaluating the effect of OxC-beta on the early stage of cytokine response and phagocytic activity (Burton et al., 2014; Johnston et al., 2014). Indirect support for the systemic availability of OxC-beta is provided in a preliminary study to be reported elsewhere, with prima facie evidence of OxC-beta activity in vivo in exploratory animal feeding trials and OxC-beta in feed-improved measures of production efficiency (i.e., feed conversion and growth), in both swine and broiler chickens (Johnston et al., 2014). Further support for the systemic availability of orally administered OxC-beta was provided in a study showing enhanced resolution of inflammation in a model of bovine respiratory disease (Duquette et al., 2014). In this study, there was no growth promotion by OxC-beta. At 14 d, the MBW of the virginiamycin-treated group was significantly higher than that of the other groups. This is presumably due to the growth promotion effect of antibiotics; the bacitracin group showed the highest MBW after the challenge, and may be because of the combined action of growth promotion and antimicrobial effects. OxC-beta may not be as promising as antibiotics in terms of growth promotion, although weight gain may vary depending on animal age, period, and dose of substance administration. Nevertheless, the positive control group showed significant weight loss post-challenge, while all groups treated with OxC-beta maintained body weight similar to that of the negative control group. Thus, productivity improvement is expected by minimizing damage in case of NE outbreak. In summary, dietary OxC-beta supplements exerted notable effects on NE lesions and decreased C. perfringens in small intestine. Administration of 2 to 6 mg/kg in-feed OxC-beta can contribute to the prevention of NE at commercial broiler chicken farms, and may also have a positive effect in improving productivity in outbreaks of NE. 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Poultry ScienceOxford University Press

Published: Sep 1, 2018

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