Essential Oils as an Alternative to Antibiotics to Reduce the Incidence and Severity of Necrotic Enteritis in Broiler Chickens: A Short Review

Due to the removal of antibiotic growth promoters (AGPs) and consumer pressure for antibiotic-free (ABF) or no antibiotics ever (NAE) poultry production, there is a need for sustainable alternatives to prevent disease in commercial poultry operations. Without AGPs, there has been a rise in diseases that were traditionally controlled by subtherapeutic levels of antibiotics in the diet. This has impacted the health of commercial poultry and has been a significant cost to poultry producers. To mitigate this, the industry has started to investigate alternatives to antibiotics to treat these forthcoming health issues, such as necrotic enteritis (NE). NE is an enteric disease caused by an over proliferation of toxigenic Clostridium perfringens (CP) in the gastrointestinal tract. Although CP is a commensal in the avian intestinal tract, dysbiosis caused by inflammation and impaired intestinal integrity facilitates uncontrolled replication of CP. Infectious agents, such as Eimeria maxima, appear to be a predominant predisposing factor that promotes NE. However, non-infectious stressors, including dietary changes, have also been associated with NE to some degree. As a result of increased pressure to restrict the use of antibiotics, there is a need for research evaluating the efficacy of alternatives, such as plant-derived essential oils, as potential tools to mitigate NE in commercial poultry flocks. The aim of this study is to review the effects of essential oils as an alternative to antibiotics to reduce the incidence and severity of necrotic enteritis in broiler chickens.

Isolation, screening and identifi cation of Bacillus spp. as direct-fed microbial candidates for aflatoxin B_1 biodegradation

Objective: To evaluate the ability of Bacillus spp. as direct-fed microbials(DFM) to biodegrade al atoxin B1(AFB1) by using an in vitro digestive model simulating in vivo conditions.Methods: Sixty-nine Bacillus isolates were obtained from intestines, and soil samples were screened by using a selective media method against 0.25 and 1.00 μg/m L of AFB1 in modii ed Czapek-Dox medium. Plates were incubated at 37 °C and observed every two days for two weeks. Physiological properties of the three Bacillus spp. candidates were characterized biochemically and by 16 S r RNA sequence analyzes for identii cation. Tolerance to acidic p H, osmotic concentrations of Na Cl, bile salts were tested, and antimicrobial sensitivity proi les were also determined. Bacillus candidates were individually sporulated by using a solid fermentation method and combined. Spores were incorporated into 1 of 3 experimental feed groups: 1) Negative control group, with unmedicated starter broiler feed without AFB1; 2) Positive control group, with negative control feed contaminated with 0.01% AFB1; 3) DFM treated group, with positive control feed supplemented with 109 spores/g. After digestion time(3:15 h), supernatants and digesta were collected for high-performance liquid chromatography l uorescence detection analysis by triplicate.Results: Three out of those sixty-nine DFM candidates showed ability to biodegrade AFB1 in vitro based on growth as well as reduction of l uorescence and area of clearance around each colony in modii ed Czapek-Dox medium which was clearly visible under day light after 48 h of evaluation. Analysis of 16S-DNA identii ed the strains as Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus subtilis. The three Bacillus strains were tolerant to acidic conditions(p H 2.0), tolerant to a high osmotic pressure(Na Cl at 6.5%), and were able to tolerate 0.037% bile salts after 24 h of incubation. No signii cant dif erences(P > 0.05) were observed in the concentrations of AFB1 in neither the supernatants nor digesta samples evaluated by highperformance liquid chromatography with l uorescence detection between positive control or DFM treated groups. Conclusions: In vitro digestion time was not enough to confirm biodegradation of AFB1. Further studies to evaluate the possible biodegradation ef ects of the BacillusDFM when continuously administered in experimentally contaminated feed with AFB1, are in progress.

Evaluation of curcumin and copper acetate against Salmonella Typhimurium infection,intestinal permeability,and cecal microbiota composition in broiler chickens

Background:Interest in the use of natural feed additives as an alternative to antimicrobials in the poultry industry has increased in recent years because of the risk of bacterial resistance.One of the most studied groups are polyphenolic compounds,given their advantages over other types of additives and their easy potentiation of effects when complexes are formed with metal ions.Therefore,the objective of the present study was to evaluate the impact of dietary supplementation of copper acetate(CA),curcumin(CR),and their combination(CA-CR)against Salmonella Typhimurium colonization,intestinal permeability,and cecal microbiota composition in broiler chickens through a laboratory Salmonella infection model.S.Typhimurium recovery was determined on day 10 post-challenge by isolating Salmonella in homogenates of the right cecal tonsil(12 chickens per group)on Xylose Lysine Tergitol-4(XLT-4)with novobiocin and nalidixic acid.Intestinal integrity was indirectly determined by the fluorometric measurement of fluorescein isothiocyanate dextran(FITC-d)in serum samples from blood obtained on d 10 post-S.Typhimurium challenge.Finally,microbiota analysis was performed using the content of the left caecal tonsil of 5 chickens per group by sequencing V4 region of 16S rRNA gene.Results:The results showed that in two independent studies,all experimental treatments were able to significantly reduce the S.Typhimurium colonization in cecal tonsils(CT,P<0.0001)compared to the positive control(PC)group.However,only CA-CR was the most effective treatment in reducing S.Typhimurium counts in both independent studies.Furthermore,the serum fluorescein isothiocyanate dextran(FITC-d)concentration in chickens treated with CR was significantly lower when compared to PC(P=0.0084),which is related to a decrease in intestinal permeability and therefore intestinal integrity.The effect of dietary treatments in reducing Salmonella was further supported by the analysis of 16S rRNA gene sequences using Linear discriminant analysis effect size(LEfSe)since Salmonella was significantly enriched in PC group(LDA score>2.0 and P<0.05)compared to other groups.In addition,Coprobacillus,Eubacterium,and Clostridium were significantly higher in the PC group compared to other treatment groups.On the contrary,Fecalibacterium and Enterococcus in CR,unknown genus of Erysipelotrichaceae at CA-CR,and unknown genus of Lachnospiraceae at CA were significantly more abundant respectively.Conclusions:CR treatment was the most effective treatment to reduce S.Typhimurium intestinal colonization and maintain better intestinal homeostasis which might be achieved through modulation of cecal microbiota.

The Effect of a<i>Lactobacillus</i>-Based Probiotic for the Control of Necrotic Enteritis in Broilers

Necrotic Enteritis (NE) caused by Clostridium perfringens (CP) in poultry is probably the most important bacterial disease in terms of economic implications. The disease is multi-factorial and is invariably associated with predisposing factors. The present study investigated the effect of a commercially available Lactobacillus-based probiotic (FM-B11) for the control of necrotic enteritis in broiler chickens. In experiment 1, one-day-of-hatch broiler chicks were randomly allocated to the following treatment groups: 1) Non-challenged (NC);2) Challenged (C);3) Challenged + probiotic (C+ FM-B11). Prior to placement, chicks in groups 2 and 3 received 0.25 mL of Salmonella typhimurium (ST) containing 105 cfu of viable cells by oral gavage. At 14, 15 and 16 days of age, all chicks in group 3 were treated with FM-B11 in the drinking water at a concentration of 106 cfu/ml. At 21d of age, all chicks in groups 2 and 3, were individually challenged with 5 × 104 sporulated oocysts of E. maxima by oral gavage. At 26d of age, all chicks in groups 2 and 3, were individually challenged with 108 cfu CP;body weight (BW) was recorded prior to challenge. The experiment was terminated at 29 days of age and the following parameters were evaluated: NE-associated mortality, CP lesion scores, CP concentrations in ileum, BW, and body weight gain (BWG). Chicks treated with FM-B11 had significantly (P < 0.05) higher body weight gain after challenge when compared to control challenge chickens. Total mortality was higher in the C group (48.8%) when compared to the C + FM-B11 (12.7%). Even though there was no significant (P > 0.05) difference in lesion score between C and C + FM-B11, group C + FM-B11 had significantly (P < 0.05) lower total number of cfu of CP recovered from the ileal mucosa and content samples when compared to group C. Experiment 2 was a unique and remarkable case report of a field outbreak of NE in a commercial broiler farm in Argentina. A reduction and control of the mortality associated with NE following 3 days of administration of FM-B11 was observed as compared with the control non treated house. These results imply that the commercially available Lactobacillus-based probiotic FM-B11 was able to reduce the severities of NE, as a secondary bacterial infection, in an experimental NE challenge model;as well as, in a commercial field outbreak of NE.

Comparison of total immunoglobulin A levels in different samples in Leghorn and broiler chickens

Objective: To standardize an ELISA protocol to quantify total immunoglobulin A(IgA)from different biological samples.Methods: Two independent experiments were conducted. In Experiment 1, total IgA levels were quantified from the lachrymal fluid, tracheal swab, and cloacal swab at various time points from Days 30 to 89 in white Leghorn chickens. Experiment 2 was conducted to evaluate the effect of 50 or 500 ppb of aflatoxin B1(AFB1) on total IgA quantified in samples from the lachrymal fluid, tracheal swab, gut content and cloacal swab in broiler chickens at 21 days of age.Results: Lachrymal fluid contained the highest level of IgA; however, the sampling procedure was time consuming and stressful to the bird, and the sample volume depends largely on the size of the chicken. Cloacal swabs also contained a high concentration of IgA; this sampling procedure was faster than lachrymal fluid sampling and was not affected by the age of the bird. Tracheal sampling was more difficult than cloacal sampling; the age of the bird limited the sampling, and the IgA concentration was the lowest detected at all sampling ages. 500 ppb of AFB1 significantly reduced total IgA concentration in the gut content compared with control or 50 ppb of AFB1 treated groups.Interestingly, a significant reduction in total IgA was also observed in those chickens that received 50 ppb of AFB1 in gut rinse when compared with cloacal swabs.Conclusions: The results of this study suggest that cloacal swab is an easy and reliable way to evaluate mucosal IgA concentration in both Leghorn and broiler chickens.

Fatty Acid Deposition on Broiler Meat in Chickens Supplemented with Tuna Oil

The aim of the present study was to evaluate the effect of feeding four levels of tuna oil on performance and fatty acid (FA) profiles of broiler chicken meat. 240 Ross broiler chickens were randomly assigned to 20 pens and divided into four treatments: 0%, 0.75%, 1%, and 1.25% of tuna oil. At 49 days, breast and legs-thighs muscles were processed for FA analysis. Concentrations of FA in legs and thighs meat were significantly higher when compared with breast meat. In both types of meats (breast and legs-thighs), the inclusion of 1.0% or 1.25% of tuna oil in the diet significantly increased (P < 0.05) the concentration of n-3 FA (especially docosahexaenoic and eicosapentaenoic acids). Fat in broiler breast contained a proportion of 29% saturated FA (SFA): 36% monosaturated FA (MFA): 35% polyunsaturated FA (PUFA);while legs and thighs meat had a proportion of 28% SFA: 38% MFA: 33% PUFA. The addition of tuna oil in the broiler diet significantly reduced the deposition of SFA, MFA, and PUFA in breast meat, while in legs and thighs these reductions were less noticeable. The significant reduction in the concentration of n-6 PUFA and the increment of n-3 PUFA were more evident in breast than in legs and thighs, and with the addition of 1% and 1.25% of tuna oil. The results of the present study suggest a difference in FA deposition attributable to supplementation with tuna oil. The addition of tuna oil could be recommendable to increase n-3 PUFA in both broiler chicken breast and legs-thighs meats, providing a healthier and functional chicken meat to consumer.

Effect of Time and Fatty Acid Composition in Eggs of White Leghorn Hens Supplemented with Tuna Oil

The study evaluated the effect of time and fatty acid (FA) composition in eggs of laying hens supplemented with tuna oil (TO). Two hundred White Leghorn hens 30-week were divided into 5 treatments with 4 replicates of 10 birds each. Treatments were assigned randomly and consisted of 0%, 0.75%, 1.50%, 2.25% and 3% TO in commercial sorghum-soybean meal diets, in which the soybean oil was partially replaced. The experiment was conducted for 90 days. At days 28, 56 and 84 of the experiment, 10 eggs per replicate were collected for chemical analysis. Feed intake, egg production rate, egg weight, and feed conversion were not influenced by dietary treatment. FA content was significantly altered (P < 0.05) by TO, showing a progressive increase in egg n-3 FA (especially docosahexaenoic [DHA] and eicosapentaenoic [EPA] acids) when TO was added. Levels of EPA and DHA were higher (P < 0.05) in the egg lipids of TO fed hens than those in the control group. This was correlated (P < 0.05) with the concentration of TO in the diet. However, no significant differences were observed either in egg weight or percent of egg lay between groups. There was a high correlation (P < 0.05) between TO inclusion level in the diet and the incorporation of EPA (r = 0.96) or DHA (r = 0.92) into the egg yolk and an overall decrease in n-6 FA. There was a high correlation (P < 0.05) between TO inclusion level and the incorporation of linoleic acid (LA, r = -0.95) or arachidonic acid (AA, r = -0.96) into the egg yolk. The highest incorporation (P < 0.05) of total n-3 FA content in eggs was obtained with 3% TO/kg. This increase was proportional to TO inclusion levels in the diets (r = 0.95). The results indicate that the n-3 FA content in eggs can be increased by dietary supplementation with TO, and that TO can serve as a reasonable alternative feed ingredient in layer diets to produce a healthier choice of egg.

Combination of a <i>Lactobacillus</i>-Based Probiotic and Organic Acids Decrease Egg to Chick Weight Loss and Reduce <i>Salmonella</i>spp. Counts in the Litter of Commercial Broiler Breeders

The effects of supplementing a blend of organic acids (OA) and a lactic acid bacteria (LAB) based-probiotic on egg to chick weight loss (%) and Salmonella spp. recovery counts in the litter of commercial broiler breeders were examined in three independent trials during the years 2013, 2014 and 2015. In each trial, ten thousand birds were divided into two groups of 5000 birds (4500 females and 500 males in each group): Control non-treated group, receiving regular water or treated group, receiving OA and probiotic in the drinking water from 25 to 35 weeks of age. During the ten weeks of evaluation, one thousand fertile eggs or hatched chickens in each trial respectively, were evaluated to obtained, hatching set weight, transferred egg weight, and hatching chick weight, to estimate the difference between egg to chick weight loss (%). Besides, in trial 2 (2014) and trial 3 (2015), litter samples were collected to evaluate Salmonella spp. counts. The supplementation of OA and probiotic during ten consecutive weeks significantly decreased the egg to chick weight loss (%) when compared with the control non-treated groups in all three trials evaluated. Interestingly, at the end of the trials 2 and 3, no counts of Salmonella spp. in the litter were detected. In contrast, control-non treated groups resulted in 4.30 and 4.24 Log10 of Salmonella spp. in trials two and three respectively. The results of the present study suggest that supplementation of OA and a LAB-probiotic for ten consecutive weeks decrease the egg to chick weight loss (%) and reduce Salmonella spp. counts in the litter of commercial broiler breeders. Higher initial body weight in broiler chickens and reduction of Salmonella spp., clearly justify the use of the combination of these products, as alternatives to antibiotics.