Search

Essential oils (EOs) have been considered as an alternative to antibiotics for animal production. In the current study, 4 trials were conducted on a commercial broiler farm to investigate the effects of dietary supplementation of an encapsulated cinnamon EO product (NE-OFF) on the bird growth performance, gut health, and gene expression in the ileum, spleen, and liver relating to the host response to heat and other stresses, including potential NE challenge. In each trial, approximately 30,000 Cobb or Ross broilers were randomly allocated to 4 treatments: a raised without antibiotics (RWA) commercial diet as positive control, an adjusted RWA commercial diet as negative control, and the negative control diet supplemented with 2 different dosages of NE-OFF, which was added during feed pelleting. Although the final average body weight did not differ significantly among treatment groups, birds fed NE-OFF had an increased ratio of villus height and crypt depth in the jejunum, and reduced fecal oocyst counts. Trial 2 was conducted in the summer and had a necrotic enteritis (NE) outbreak. The supplementation of NE-OFF reduced the NE incidence and bird mortality. The samples from Trial 2 were hence selected for the analyses of Clostridium perfringens and NetB toxin gene abundance in the ileum, and host responses. The C. perfringens population appeared to be positively correlated with the NetB gene abundance. The gene expression analysis suggested that NE-OFF supplementation improved nutrient absorption and transportation as well as antioxidant activities to help the birds against stress. These on-farm trial results support the hypothesis that the use of NE-OFF as a feed additive can improve bird gut health and performance in commercial broiler production, especially for preventing NE outbreaks when birds are under stress., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Black soldier fly larvae meal (BSFLM) is receiving great attention as a rich source of protein and antimicrobials for poultry. Therefore, we evaluated the effects of partially or completely replacing soybean meal (SBM) with commercial BSFLM on growth performance, tibia traits, cecal short chain fatty acid (SCFA) concentrations, and excreta metabolomes in broiler chickens (Gallus gallus domesticus). A total of 480 day-old male Ross × Ross 708 chicks were assigned to 6 diets (8 replicates/diet): a basal corn-SBM diet with in-feed bacitracin methylene disalicylate (BMD), a corn-SBM diet without BMD (0% BSFLM), and four diets in which the SBM was substituted with 12.5, 25, 50, and 100% BSFLM. Body weight (BW), feed intake (FI) and cumulative feed conversion ratio (cFCR) were monitored on days 14, 28, and 35. Cecal SCFA levels were determined on days 14, 28, and 35. Tibia traits and excreta metabolomes were determined on day (d) 35. On d14, birds fed 12.5 and 25% BSFLM had a similar BW, FI, and cFCR as birds fed BMD (P > 0.05). On d 35, birds fed 12.5% BSFLM had a similar BW, FI and cFCR as birds fed BMD or 0% BSFLM (P > 0.05). For each phase, birds fed 100% BSFLM had a lower BW, FI and higher cFCR than birds fed BMD or 0% BSFLM (P < 0.05). On d 35, BW decreased linearly, quadratically, and cubically with increasing levels of BSFLM (P < 0.01). Overall (d 0-35), BSFLM linearly, quadratically, and cubically decreased FI and quadratically and cubically increased cFCR (P < 0.01). Quadratic responses were observed for tibia fresh weight (P = 0.049) and ash content (P = 0.022). BSFLM did not impact cecal SCFAs levels. The excreta metabolome of birds fed 100% BSFLM clustered independently from all other groups and exhibited greater levels of putatively identified methionine, lysine, valine, glutamine, histidine and lower levels of arginine as compared to all diets. Taken together, substitution of SBM with ≤25% of BSFLM in the starter phase may be used as an alternative to BMD., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

This study evaluated effects of feeding low-bush wild blueberry (LBP) and organic American cranberry (CRP) pomaces without or with multienzyme supplement (ENZ) on growth performance, organ weight and plasma metabolites in broiler chickens. Nonenzyme-fed (no-ENZ: n = 1,575) and enzyme-fed (ENZ: n = 1,575) day-old male Cobb500 broilers were placed in floor pens (45 chicks/pen) and allocated to five corn-soybean meal-based diets: a basal diet supplemented with either bacitracin methylene disalicylate (BMD, 55 mg/kg), 0.5 or 1% of CRP or LBP in a 2 × 5 factorial design for 35-day experiment. Body weight (BW), feed intake (FI) and mortality were recorded whereas BW gain (BWG) and feed conversion ratio (FCR) were calculated. Birds were sampled at days 21 and 35 for organ weights and plasma metabolites. There were no interactions between diet and ENZ on any parameter (P > 0.05) and no effect of ENZ on overall (d 0-35) growth performance and organ weights (P > 0.05). Birds fed BMD were heavier (P < 0.05) at d 35 and had better overall FCR than berry-supplemented birds. Birds fed 1% LBP had poor FCR than birds fed 0.5% CRP. Birds fed LBP exhibited heavier liver (P < 0.05) than birds fed BMD or 1% CRP. The highest plasma concentrations of aspartate transaminase (AST), creatine kinase (CK) at d 28 and gamma-glutamyl transferase (GGT) at d 35 were found in ENZ-fed birds (P < 0.05). At d 28, birds fed 0.5% LBP showed higher plasma AST and CK concentrations (P < 0.05). However, CRP feeding resulted in a lower plasma CK level compared with BMD feeding (P < 0.05). The lowest cholesterol level was detected in 1% CRP-fed birds. In conclusion, this study showed no ENZ effects to potentiate berry pomaces on the overall growth performance of broilers (P < 0.05. However, plasma profiles revealed the potential of ENZ to modulate the metabolism of pomace-fed broilers. LBP increased BW during the starter phase, while CRP increased BW during the grower phase., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

This study examined changes in soil bacterial community composition and diversity in response to fertilization with litter from chickens fed a diet without antibiotics and with bambermycin, penicillin, bacitracin, salinomycin, or mix of salinomycin and bacitracin. Litter (27.5 T/ha) was applied to 24 agricultural plots in the Fraser Valley of British Columbia. Nonfertilized plots were used as a negative control. Soil samples collected from the studied plots were used to quantify Escherichia coli by plate counts, and Clostridium perfringens by qPCR. The 16S rRNA gene sequencing was performed for microbiota analysis. Following litter application in December, the population size of E. coli was 5.4 log CFU/g; however, regardless of treatments, the results revealed 5.2 and 1.4 log CFU/g of E. coli in soil sampled in January and March, respectively. Fertilization with litter from antibiotic-treated birds increased ( P  < 0.05) the relative abundance of Proteobacteria, Actinobacteria, and Firmicutes in soil, but decreased Acidobacteria and Verrucomicrobia groups. The alpha diversity parameters were higher ( P  < 0.05) in nonfertilized soil compared to the fertilized ones, suggesting that litter application was a major factor in shaping the soil bacterial communities. These results may help develop efficient litter management strategies like composting, autoclaving, or anaerobic digestion of poultry litter before application to land for preservation of soil health and crop productivity.

Abstract: Extraintestinal pathogenic Escherichia coli (ExPEC) include several serotypes that have been associated with colibacillosis in poultry and with urinary tract infections (UTIs) and newborn meningitis in humans. In this study, 57 antimicrobial-resistant E. coli from apparently healthy broiler chickens were characterized for their health and safety risks. These isolates belonged to 12 serotypes, and isolates of the same serotype were clonal based on single nucleotide variant analysis. Most of the isolates harbored plasmids; IncC and IncFIA were frequently detected. The majority of the resistant isolates harbored plasmid-mediated resistance genes, including aph(3″)-Ib, aph(6)-Id, blaCMY-2, floR, sul1, sul2, tet(A), and tet(B), in agreement with their resistant phenotypes. The class 1 integron was detected in all E. coli serotypes except O124:H25 and O7:H6. Of the 57 broiler E. coli isolates, 27 were avian pathogenic, among which 18 were also uropathogenic E. coli and the remainder were other ExPEC. The two isolates of serotype O161:H4 (ST117) were genetically related to the control avian pathogenic strains and a clinical isolate associated with UTIs. A strain of serotype O159:H45 (ST101) also was closely related to a UTI isolate. The detected virulence factors included adhesins, invasins, siderophores, type III secretion systems, and toxins in combination with other virulence determinants. A broiler isolate of serotype O7:H18 (ST38) carried the ibeA gene encoding a protein involved in invasion of brain endothelium on a 102-kbp genetic island. This isolate moderately adhered and invaded Caco-2 cells and induced mortality (42.5%) in a day-old-chick infection model. The results of this study suggest that multiple antimicrobial-resistant E. coli isolates recovered from apparent healthy broilers can be pathogenic and act as reservoirs for antimicrobial resistance genes, highlighting the necessity of their assessment in a "One-Heath" context., (Published 2022 by the International Association for Food Protection. Copyright ©, Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada.)

Feeding practices have been found to influence gut microbiota which play a major role in immunity of poultry. In the present study, changes in cecal microbiota and humoral responses resulting in the 55 ppm bacitracin (BACI), 1% each of cranberry (CP1) and wild blueberry (BP1) pomace alone or in combination (CP+BP) feeding in broiler Cobb 500 vaccinated or not against coccidiosis were investigated. In the non-vaccinated group, no significant treatment effects were observed on performance parameters. Vaccination significantly affected bird's performance parameters particularly during the growing phase from 10 to 20 days of age. In general, the prevalence of coccidiosis and necrotic enteritis (NE) was reduced by vaccination ( P < 0.05). BACI-treated birds showed low intestinal lesion scores, and both CP1 and BP1 feed supplementations reduced Eimeria acervulina and Clostridium perfringens incidences similar to BACI. Vaccination induced change in serum enzymes, minerals, and lipid levels in 21-day old birds while, levels of triglyceride (TRIG) and non-esterified fatty acids (NEFA) were higher ( P < 0.05) in CP1 treated non-vaccinated group than in the control. The levels of NEFA were lower in BACI- and CP1-fed birds than in the control in non-vaccinated day 28 old birds. The highest levels of all estimated three immunoglobulins (IgY, IgM, and IgA) were found in the vaccinated birds. Metagenomics analysis of the cecal bacterial community in 21-day old birds showed the presence of Firmicutes (90%), Proteobacteria (5%), Actinobacteria (2%), and Bacteroidetes (2%). In the vaccinated group, an effect of BACI was noted on Proteobacteria ( P = 0.03). Vaccination and/or dietary treatments influenced the population of Lactobacillaceae , Enterobacteriaceae, Clostridiaceae , and Streptococcaceae which were among the most abundant families. Overall, this study revealed that besides their beneficial effects on performance, alike bacitracin, berry pomaces in poultry feed have profound impacts on the chicken cecal microbiota and blood metabolites that could be influenced by vaccination against coccidiosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada.)

The impact of antibiotic use for growth promotion in livestock and poultry production on the rise of antimicrobial resistance (AMR) in bacteria led to the ban of this practice in the European Union in 2006 and a restriction of antimicrobial use (AMU) in animal agriculture in Canada and the United States of America. There is a high risk of infectious diseases such as necrotic enteritis due to Clostridium perfringens , and colibacillosis due to avian pathogenic Escherichia coli in antimicrobial-free broiler chickens. Thus, efficient and cost-effective methods for reducing AMU, maintaining good poultry health and reducing public health risks (food safety) are urgently needed for poultry production. Several alternative agents, including plant-derived polyphenolic compounds, have been investigated for their potential to prevent and control diseases through increasing poultry immunity. Many studies in humans reported that plant flavonoids could modulate the immune system by decreasing production of pro-inflammatory cytokines, T-cell activation, and proliferation. Fruits, especially berries, are excellent sources of flavonoids while being rich in nutrients and other functionally important molecules (vitamins and minerals). Thus, fruit byproducts or wastes could be important resources for value-added applications in poultry production. In the context of the circular economy and waste reduction, this review summarizes observed effects of fruit wastes/extracts on the general health and the immunity of poultry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Patrick Boerlin, Shayan Sharif and Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada for the contribution of Moussa S. Diarra and Teri-Lyn Hasted.)

With the pressure to reduce antibiotics use in poultry production, cost-effective alternative products need to be developed to enhance the bird's immunity. The present study evaluated the efficacy of cranberry fruit by-products to modulate immunity in broiler chickens. Broiler Cobb 500 chicks were fed a control basal diet, basal diet supplemented with bacitracin (BACI, 55 ppm), cranberry pomace at 1% and 2% (CP2), or cranberry pomace ethanolic extract at 150 and 300 ppm (COH300) for 30 d. Blood sera were analyzed at days 21 and 28 of age for Ig levels by ELISA. The innate and adaptive immune-related gene expression levels in the liver and bursa of Fabricius were investigated at 21 d of age by quantitative polymerase chain reaction arrays. At day 21, the highest IgY level was found in the blood serum of the CP2-fed birds. In the liver, 13 of the 22 differentially expressed genes were downregulated across all treatments compared with the control. Expression of genes belonging to innate immunity such as caspase 1 apoptosis-related cysteine peptidase, chemokine receptor 5, interferon gamma, myeloid differentiation primary response gene 88, and Toll-like receptor 3 were significantly downregulated mainly in BACI- and COH300-fed birds. In the bursa, 5 of 9 genes associated with the innate immunity were differentially expressed. The expression of anti-inflammatory IL-10 gene was upregulated in all treatment groups in bursa compared with the control. The expression of transferrin gene was significantly upregulated in livers of birds fed COH300 and in bursa of birds fed BACI, indicating feeding practices and organ-dependant modulation of this gene in broiler. Overall results of this study showed that cranberry product feed supplementation modulated the innate immune and suppressed proinflammatory cytokines in broilers, providing a platform for future investigations to develop berry products in poultry feeding., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

1. A total of 864 d old (male) Ross × Ross 708 broiler chicks were allocated to 48 floor pens (12 pens/treatment and 18 birds/pen) to investigate dose-response of a blend of seaweeds (SB) on growth performance, breast yield, jejunal histomorphology, microbial metabolites and community and plasma biochemical profile. 2. A maize-soybean meal diet was formulated with 0, 5, 10 or 20 g/kg of SB. Diets were formulated for a three-phase feeding programme (starter: d 0-10, grower: d 11-24, and finisher: d 25-42) and met or exceeded Aviagen nutrient specifications. Diets were allocated to pens (n = 12) balanced for body weight (BW). Birds had free access to feed and water, BW and feed intake (FI) were monitored by phase. One bird per pen was randomly selected on d 42, bled for plasma, and samples for intestinal tissue and caecal digesta were taken. Microbial DNA was extracted and submitted for microbial community profile using the Illumina Miseq® platform. 3. In the starter phase, SB linearly ( P ≤ 0.01) improved BW, body weight gain (BWG), and FCR. However, the improvement was quadratic, such that there was no further improvement beyond 5 g/kg SB inclusion. Growth performance response to SB in the grower phase was similar to the starter phase, with the exception of FCR ( P > 0.05). Overall, from d 0-42, a linear and quadratic ( P < 0.01) response was observed for final BW (d 42), whereby birds fed 5, 10 and 20 g/kg SB were heavier than control by 166, 183 and 180 g, respectively. A quadratic ( P = 0.03) effect was observed for breast yield in response to SB. There was a quadratic reduction ( P < 0.05) in gamma-glutamyl transferase (GGT) and a linear increase in glutamate dehydrogenase (GDH) in response to SB. Supplemental SB linearly reduced ( P ≤ 0.04) the relative abundance of phylum Bacteroidetes and Proteobacteria , and increased the abundance of phylum Firmicutes (linearly; P = 0.02) and Actinobacteria (quadratically; P = 0.03). 4. The data indicated that the optimal inclusion for SB was between 5 and 10 g/kg for improved growth performance and breast yield. However, increased abundance of Firmicutes and actinobacteria in the caecal digesta suggested that the higher doses enhanced prebiotic effects of seaweed components.

This study investigated the effects of encapsulated cinnamaldehyde (CIN) and citral (CIT) alone or in combination (CIN + CIT) on the growth performance and cecal microbiota of nonvaccinated broilers and broilers vaccinated against coccidiosis. Vaccinated (1,600) and nonvaccinated (1,600) 0-day-old male Cobb500 broilers were randomly allocated to 5 treatments: basal diet (control) and basal diet supplemented with bacitracin (BAC, 55 ppm), CIN (100 ppm), CIT (100 ppm), and CIN (100 ppm) + CIT (100 ppm). In general, body weight (BW) and feed conversion ratio were significantly improved in birds treated with BAC, CIN, CIT, and CIN + CIT (P < 0.05) but were all decreased in vaccinated birds compared with nonvaccinated birds (P < 0.05). Significant interactions (P < 0.05) between vaccination and treatments for average daily gain during the periods of starter (day 0-9) and BW on day 10 were noted. Broilers receiving vaccines (P < 0.01) or feed supplemented with BAC, CIN, CIT, or CIN + CIT (P < 0.01) showed reductions in mortality rate from day 0 to 28. The incidences of minor coccidiosis were higher (P < 0.05) in vaccinated birds than in nonvaccinated birds. Diet supplementation with BAC or tested encapsulated essential oils showed comparable effects on the coccidiosis incidences. Similar to BAC, CIN and its combination with CIT reduced both incidence and severity of necrotic enteritis (P < 0.05). No treatment effects were observed on the cecal microbiota at the phyla level. At the genus level, significant differences between vaccination and treatment groups were observed for 5 (Lactobacillus, Ruminococcus, Faecalibacterium, Enterococcus, and Clostridium) of 40 detected genera (P < 0.05). The genus Lactobacillus was more abundant in broilers fed with CIT, while Clostridium and Enterococcus were less abundant in broilers fed with CIN, CIT, or CIN + CIT in both the vaccinated and nonvaccinated groups. Results from this study suggested that CIN alone or in combination with CIT in feed could improve chicken growth performance to the level comparable with BAC and alter cecal microbiota composition., (Copyright © 2019. Published by Elsevier Inc.)

There is a need to develop cost-effective approaches to modulate gut microbiota, promote bird health, and prevent infections in pasture-raised broiler chickens. The present study evaluated the efficacy of organic wild blueberry (Vaccinium angustifolium) also called low-bush blueberry pomace (LBBP)-supplemented feed to modulate the chicken gut microbiota, and blood metabolites in order to improve bird health and productivity. Slow-growing broiler chickens were reared on pasture up to 64 D for sampling after 2 wk of treatment during brooding with 0, 1, and 2% LBBP in feed. Intestinal samples were collected at different time-points throughout the trial for bacterial culture and microbial community analysis by 16S rRNA gene sequencing using Illumina MiSeq. Blood sera were also analyzed for metabolites at each sampling time. Of the 14 bacterial phyla, the predominant taxa across all sampling time-points were Firmicutes, Proteobacteria, Bacteroidetes, and Tenericutes, representing >97% of all sequences. Bacteroidetes seemed to be replacing Firmicutes by LBBP supplementation, with the most noticeable effect at day 64 with 1% LBBP. LBBP inclusion enriched Lactobacillus, Bacteroides, and Bifidobacterium, while Escherichia coli, Clostridium&#95;Clostridiaceae, Helicobacter, and Enterococcus showed higher abundances in control birds at the end of trial. Principal co-ordinate analysis showed a clear clustering of the intestinal samples from control and LBBP-treated groups at day 29. Application of LBBP resulted in a decrease (P < 0.05) in cholesterol at day 21, and an increase (P < 0.05) in high-density lipoprotein cholesterol in 14-day-old broilers. Higher (P < 0.05) levels of phosphorus, magnesium, and globulin at day 21 as well as iron and albumin at day 36 were also observed in 1% LBBP-fed birds. Despite limitations consisting essentially of low sampled birds for measurements, this study indicated that dietary supplementation of LBBP could positively influence gut microbiota and blood metabolites that may contribute to the overall health of pasture-raised broiler chickens., (© Her Majesty the Queen in the Right of Canada, as represented by the minister of Agriculture and Agri-Food Canada.)

Studies on effects of cranberry products in animals, especially in chickens, are very scarce or even lacking. This study investigated the immunomodulatory effect of high molecular weight non-dialyzable materials (NDMs) of cranberry extract. The in vitro antioxidant and anti-inflammatory activities were investigated. The ability of NDMs (0, 1, 2, or 4 mg/mL) to enhance phagocytic activities was also evaluated using chicken heterophils (CHEs) against Staphylococcus aureus Furthermore, a broiler model was used to determine the effect of NDMs on the humoral immune response. Seven-d-old chicks were vaccinated with the infectious bursal disease virus (IBDV) vaccine S-706, and treated orally with 0, 2, 4, or 8 mg/mL/bird NDMs for five consecutive days. Serum immunoglobulin level (Ig), and antibody concentration against IBDV, infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and avian reovirus (ARV) were measured weekly by enzyme-linked immunosorbent assay (ELISA). NDMs showed >five-fold higher antioxidant activity (oxygen radical absorbance capacity = 222.7 mg trolox/g) than the commercial raw cranberry juice from which it derived (oxygen radical absorbance capacity = 39.6 mg trolox/g). Likewise, NDMs demonstrated anti-inflammatory activities comparable to Naproxen but better than those of Ibuprofen. The susceptibility of S. aureus to phagocytosis by CHEs increased significantly (P < 0.05) at 4 mg/mL NDMs in the medium. While no intracellular bacteria were counted in CHEs after phagocytosis in the presence of 2 and 4 mg/mL NDMs, 1 mg/mL NDMs demonstrated a significant (P < 0.05) intracellular killing activity in CHEs against S. aureus compared to the untreated CHEs. Results from the in vivo studies indicated that birds receiving 2 and 4 mg/mL/bird NDMs had a higher serum IgM level (P < 0.05), and their antibody titers against IBDV tended to increase with NDMs administration (P = 0.06) on d 35. These results suggest that NDMs enhances bacterial susceptibility to immuno-defense mechanisms, and may be useful as immunomodulators against infections., (© Crown copyright 2016.)

The objective of this study was to investigate the antibiotic resistance of Escherichia fergusonii isolated from commercial broiler chicken farms. A total of 245 isolates from cloacal and cecal samples of 28- to 36-day-old chickens were collected from 32 farms. Isolates were identified using PCR, and their susceptibility to 16 antibiotics was determined by disk diffusion assay. All isolates were susceptible to meropenem, amikacin, and ciprofloxacin. The most common resistances were against ampicillin (75.1%), streptomycin (62.9%), and tetracycline (57.1%). Of the 184 ampicillin-resistant isolates, 127 were investigated using a DNA microarray carrying 75 probes for antibiotic resistance genetic determinants. Of these 127 isolates, the β-lactamase blaCMY2, blaTEM, blaACT, blaSHV, and blaCTX-M-15 genes were detected in 120 (94.5%), 31 (24.4%), 8 (6.3%), 6 (4.7%), and 4 (3.2%) isolates, respectively. Other detected genes included those conferring resistance to aminoglycosides (aadA1, strA, strB), trimethoprims (dfrV, dfrA1), tetracyclines (tetA, tetB, tetC, tetE), and sulfonamides (sul1, sul2). Class 1 integron was found in 35 (27.6%) of the ampicillin-resistant isolates. However, our data showed that the tested E. fergusonii did not carry any carbapenemase blaOXA genes. Pulsed-field gel electrophoresis revealed that the selected ampicillin-resistant E. fergusonii isolates were genetically diverse. The present study indicates that the monitoring of antimicrobial-resistant bacteria should include enteric bacteria such as E. fergusonii, which could be a reservoir of antibiotic resistance genes. The detection of isolates harboring extended-spectrum β-lactamase genes, particularly blaCTX-M-15, in this work suggests that further investigations on the occurrence of such genes in broilers are warranted.

There is an urgent need to control necrotic enteritis (NE) caused by Clostridium perfringens in chickens when antibiotics are withdrawn from feed. Carvacrol has strong antimicrobial activity and its delivery to the animal intestine can be significantly enhanced after encapsulation. The present study has investigated the potential of encapsulated carvacrol in controlling NE. In general, micro-encapsulation of carvacrol in an alginate-whey protein matrix showed no adverse effect on its antimicrobial activity towards C. perfringens in either Brain Heart Infusion (BHI) broth or a simulated gastrointestinal model. The minimum inhibitory concentrations of both encapsulated and un-encapsulated carvacrol were approximately 200 μl/l against C. perfringens in BHI. In a broiler infection model with C. perfringens, the diets supplemented with encapsulated carvacrol at the dose of either 250 or 650 μg/g significantly reduced NE in the chicken intestine, which was close to the degree of lesions observed in bacitracin/salinomycin treated birds. Supplementation with either bacitracin/salinomycin or encapsulated carvacrol showed no significant impact on intestinal burden of Lactobacillus. However, the treatment with bacitracin/salinomycin or the low dose of encapsulated carvacrol reduced the level of C. perfringens in the ileum of birds at 35 days of age. These results suggest that our encapsulated carvacrol can be used to combat NE disease in chickens.

Background: Non-typhoidal Salmonella enterica serovars, associated with different foods including poultry products, are important causes of bacterial gastroenteritis worldwide. The colonization of the chicken gut by S. enterica could result in the contamination of the environment and food chain. The aim of this study was to compare the genomes of 25 S. enterica serovars isolated from broiler chicken farms to assess their intra- and inter-genetic variability, with a focus on virulence and antibiotic resistance characteristics., Methodology/principal Finding: The genomes of 25 S. enterica isolates covering five serovars (ten Typhimurium including three monophasic 4,[5],12:i:, four Enteritidis, three Hadar, four Heidelberg and four Kentucky) were sequenced. Most serovars were clustered in strongly supported phylogenetic clades, except for isolates of serovar Enteritidis that were scattered throughout the tree. Plasmids of varying sizes were detected in several isolates independently of serovars. Genes associated with the IncF plasmid and the IncI1 plasmid were identified in twelve and four isolates, respectively, while genes associated with the IncQ plasmid were found in one isolate. The presence of numerous genes associated with Salmonella pathogenicity islands (SPIs) was also confirmed. Components of the type III and IV secretion systems (T3SS and T4SS) varied in different isolates, which could explain in part, differences of their pathogenicity in humans and/or persistence in broilers. Conserved clusters of genes in the T3SS were detected that could be used in designing effective strategies (diagnostic, vaccination or treatments) to combat Salmonella. Antibiotic resistance genes (CMY, aadA, ampC, florR, sul1, sulI, tetAB, and srtA) and class I integrons were detected in resistant isolates while all isolates carried multidrug efflux pump systems regardless of their antibiotic susceptibility profile., Conclusions/significance: This study showed that the predominant Salmonella serovars in broiler chickens harbor genes encoding adhesins, flagellar proteins, T3SS, iron acquisition systems, and antibiotic and metal resistance genes that may explain their pathogenicity, colonization ability and persistence in chicken. The existence of mobile genetic elements indicates that isolates from a given serovar could acquire and transfer genetic material. Conserved genes in the T3SS and T4SS that we have identified are promising candidates for identification of diagnostic, antimicrobial or vaccine targets for the control of Salmonella in broiler chickens.

Escherichia fergusonii is an emerging pathogen that has been isolated from a wide range of infections in animals and humans. Primers targeting specific genes, including yliE (encoding a conserved hypothetical protein of the cellulose synthase and regulator of cellulose synthase island), EFER&#95;1569 (encoding a hypothetical protein, putative transcriptional activator for multiple antibiotic resistance), and EFER&#95;3126 (encoding a putative triphosphoribosyl-dephospho-coenzyme A [CoA]), were designed for the detection of E. fergusonii by conventional and real-time PCR methods. Primers were screened by in silico PCR against 489 bacterial genomic sequences and by both PCR methods on 55 reference and field strains. Both methods were specific and sensitive for E. fergusonii, showing amplification only for this bacterium. Conventional PCR required a minimum bacterial concentration of approximately 10(2) CFU/ml, while real-time PCR required a minimum of 0.3 pg of DNA for consistent detection. Standard curves showed an efficiency of 98.5%, with an R(2) value of 0.99 for the real-time PCR assay. Cecal and cloacal contents from 580 chickens were sampled from broiler farms located in the Fraser Valley (British Columbia, Canada). Presumptive E. fergusonii isolates were recovered by enrichment and plating on differential and selective media. Of 301 total presumptive isolates, 140 (46.5%) were identified as E. fergusonii by biochemical profiling with the API 20E system and 268 (89.0%) using PCR methods. E. fergusonii detection directly from cecal and cloacal samples without preenrichment was achieved with both PCR methods. Hence, the PCR methods developed in this work significantly improve the detection of E. fergusonii.

The objective of this study was to analyze the antibiotic resistance phenotype and genotype of Salmonella isolated from broiler production facilities. A total of 193 Salmonella isolates recovered from commercial farms in British Columbia, Canada, were evaluated. Susceptibility to antibiotics was determined with the Sensititre system. Virulence and antibiotic resistance genes were detected by PCR assay. Genetic diversity was determined by pulse-field gel electrophoresis (PFGE) typing. Seventeen serovars of Salmonella were identified. The most prevalent Salmonella serovars were Kentucky (29.0% of isolates), Typhimurium (23.8%), Enteritidis (13.5%), and Hadar (11.9%); serovars Heidelberg, Brandenburg, and Thompson were identified in 7.7, 4.1, and 3.6% of isolates, respectively. More than 43% of the isolates were simultaneously resistant to ampicillin, amoxicillin-clavulanic acid, ceftiofur, cefoxitim, and ceftriaxone. This β-lactam resistance pattern was observed in 33 (58.9%) of the Salmonella Kentucky isolates; 2 of these isolates were also resistant to chloramphenicol, streptomycin, sulfisoxazole, and tetracycline. Genes associated with resistance to aminoglycosides (aadA1, aadA2, and strA), β-lactams (blaCMY-2, blaSHV, and blaTEM), tetracycline (tetA and tetB), and sulfonamide (sul1) were detected among corresponding resistant isolates. The invasin gene (invA) and the Salmonella plasmid virulence gene (spvC) were found in 97.9 and 25.9% of the isolates, respectively, with 33 (71.7%) of the 46 Salmonella Typhimurium isolates and 17 (65.4%) of the 26 Salmonella Enteritidis isolates carrying both invA and spvC. PGFE typing revealed that the antibiotic-resistant serovars were genetically diverse. These data confirm that broiler chickens can be colonized by genetically diverse antibiotic-resistant Salmonella isolates harboring virulence determinants. The presence of such strains is highly relevant to food safety and public health.

In an effort to explore strategies to control Clostridium perfringens, we investigated the synergistic effect of a ubiquitous bacterial second messenger 3',5'-cyclic diguanylic acid (c-di-GMP) with penicillin G in a broiler challenge model. All chicks were inoculated in the crop by gavage on d 14, 15, and 16 with a mixture of 4 C. perfringens strains. Birds were treated with saline (control group) or 20 nmol of c-di-GMP by gavage or intramuscularly (IM) on d 24, all in conjunction with penicillin G in water for 5 d. Weekly samplings of ceca and ileum were performed on d 21 to 35 for C. perfringens and Lactobacillus enumeration. On d 35 of age, the IM treatment significantly (P < 0.05) reduced C. perfringens in the ceca, suggesting possible synergistic activity between penicillin G and c-di-GMP against C. perfringens in broiler ceca. Moreover, analysis of ceca DNA for the presence of a series of C. perfringens virulence genes showed a prevalence of 30% for the Clostridium perfringens alpha-toxin gene (cpa) from d 21 to 35 in the IM-treated group, whereas the occurrence of the cpa gene increased from 10 to 60% in the other 2 groups (control and gavage) from d 21 to 35. Detection of β-lactamase genes (blaCMY-2, blaSHV, and blaTEM) indicative of gram-negative bacteria in the same samples from d 21 to 35 did not show significant treatment effects. Amplified fragment-length polymorphism showed a predominant 92% similarity between the ceca of 21-d-old control birds and the 35-d-old IM-treated c-di-GMP group. This suggests that c-di-GMP IM treatment might be effective at restoring the normal microflora of the host on d 35 after being challenged by C. perfringens. Our results suggest that c-di-GMP can reduce the colonization of C. perfringens in the gut without increasing the selection pressure for some β-lactamase genes or altering the commensal bacterial population.

An Escherichia spp. isolate, ECD-227, was previously identified from the broiler chicken as a phylogenetically divergent and multidrug-resistant Escherichia coli possessing numerous virulence genes. In this study, whole genome sequencing and comparative genome analysis was used to further characterize this isolate. The presence of known and putative antibiotic resistance and virulence open reading frames were determined by comparison to pathogenic (E. coli O157:H7 TW14359, APEC O1:K1:H7, and UPEC UTI89) and nonpathogenic species (E. coli K-12 MG1655 and Escherichia fergusonii ATCC 35469). The assembled genome size of 4.87 Mb was sequenced to 18-fold depth of coverage and predicted to contain 4,376 open reading frames. Phylogenetic analysis of 537 open reading frames present across 110 enteric bacterial species identifies ECD-227 to be E. fergusonii. The genome of ECD-227 contains 5 plasmids showing similarity to known E. coli and Salmonella enterica plasmids. The presence of virulence and antibiotic resistance genes were identified and localized to the chromosome and plasmids. The mutation in gyrA (S83L) involved in fluoroquinolone resistance was identified. The Salmonella-like plasmids harbor antibiotic resistance genes on a class I integron (aadA, qacEΔ-sul1, aac3-VI, and sulI) as well as numerous virulence genes (iucABCD, sitABCD, cib, traT). In addition to the genome analysis, the virulence of ECD-227 was evaluated in a 1-d-old chick model. In the virulence assay, ECD-227 was found to induce 18 to 30% mortality in 1-d-old chicks after 24 h and 48 h of infection, respectively. This study documents an avian multidrug-resistant and virulent E. fergusonii. The existence of several resistance genes to multiple classes of antibiotics indicates that infection caused by ECD-227 would be difficult to treat using antimicrobials currently available for poultry.

A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA, recA, pheS, and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium, E. faecalis, E. durans, E. gallinarum, and E. avium. The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae. Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential.

Cranberry fruit components have been reported to have antimicrobial activities against a variety of pathogenic bacteria and to be beneficial for human health. Studies on their effects are very limited in animals and especially in chickens. This study investigated the effect of feed supplementation with a commercial cranberry fruit extract (CFE) on the performance, breast meat quality, and intestinal integrity of broiler chickens. Twelve hundred male 1-d-old broiler chicks were allocated randomly to CFE treatments at 0, 40, 80, or 160 mg/kg of feed from d 0 to 35. Cloacal and cecal samples were collected weekly to evaluate the influence of treatments on the intestinal population of generic Escherichia coli, Clostridium perfringens, Enterococcus spp., and Lactobacillus spp. At d 35, BW were 1.62, 1.60, 1.61, and 1.64 kg for the control birds and birds fed 40, 80, and 160 mg of CFE/kg of feed, respectively. Feed intake ranged from 2.7 to 2.8 kg and feed efficiency from 1.8 to 1.9 g of feed/g of BW. However, the treatment effects on bird performance were not statistically significant (P > 0.05). The mortality rate tended to be lower (P = 0.09) in birds fed 40 mg of CFE/kg of feed. Feed supplementation with CFE did not significantly alter any broiler meat properties evaluated when compared with the control diet (P > 0.05). At d 28, the populations of Enterococcus spp. in cecal and cloacal samples were significantly lower (P < 0.05) in birds receiving CFE at 160 mg/kg of feed than the other groups. No significant differences were noted between the control and the treatment groups for general health and intestinal integrity (P > 0.05). These findings suggest that more studies are needed to investigate potential beneficial effects of CFE or its derivatives in broiler production.

Veterinary pharmaceuticals are commonly used in poultry farming to prevent and treat microbial infections as well as to increase feed efficiency, but their use has created public and environmental health concerns. Poultry litter contains antimicrobial residues and resistant bacteria; when applied as fertilizer, the level and effects of these pharmaceuticals and antimicrobial-resistant bacteria in the environment are of concern. The purpose of this study was to investigate poultry litter for veterinary pharmaceuticals and resistance patterns of Escherichia coli. Litter samples were collected from controlled feeding trials and from commercial farms. Feed additives bacitracin, chlortetracycline, monensin, narasin, nicarbazin, penicillin, salinomycin, and virginiamycin, which were present in the feed on commercial farms and added to the feed in the controlled trials, were extracted in methanol and analyzed by liquid chromatography-mass spectrometry techniques. Sixty-nine E. coli were isolated and identified by API 20E. The susceptibility of the isolates to antibiotics was determined using Avian plates and the Sensititer automated system. This study confirmed the presence of antimicrobial residues in broiler litter from controlled environments as well as commercial farms, ranging from 0.07 to 66 mg/L depending on the compound. Concentrations of individual residues were higher in litter from controlled feeding trials than those from commercial farms. All E. coli isolates from commercial farms were multiresistant to at least 7 antibiotics. Resistance to beta-lactam antibiotics (amoxicillin, ceftiofur), tetracyclines, and sulfonamides was the most prevalent. This study concluded that broiler litter is a source of antimicrobial residues and represents a reservoir of multiple antibiotic-resistant E. coli.

The impact of feed supplementation with bambermycin, monensin, narasin, virginiamycin, chlortetracycline, penicillin, salinomycin, and bacitracin on the distribution of Escherichia coli pathotypes in broiler chickens was investigated using an E. coli virulence DNA microarray. Among 256 E. coli isolates examined, 59 (23%) were classified as potentially extraintestinal pathogenic E. coli (ExPEC), while 197 (77%) were considered commensal. Except for chlortetracycline treatment, the pathotype distribution was not significantly different among treatments (P > 0.05). Within the 59 ExPEC isolates, 44 (75%) were determined to be potentially avian pathogenic E. coli (APEC), with the remaining 15 (25%) considered potentially "other" ExPEC isolates. The distribution within phylogenetic groups showed that 52 (88%) of the ExPEC isolates belonged to groups B2 and D, with the majority of APEC isolates classified as group D and most commensal isolates (170, 86%) as group A or B1. Indirect assessment of the presence of the virulence plasmid pAPEC-O2-ColV showed a strong association of the plasmid with APEC isolates. Among the 256 isolates, 224 (88%) possessed at least one antimicrobial resistance gene, with nearly half (107, 42%) showing multiple resistance genes. The majority of resistance genes were distributed among commensal isolates. Considering that the simultaneous detection of antimicrobial resistance tet(A), sulI, and bla(TEM) genes and the integron class I indicated a potential presence of the resistance pAPEC-O2-R plasmid, the results revealed that 35 (14%) of the isolates, all commensals, possessed this multigene resistance plasmid. The virulence plasmid was never found in combination with the antimicrobial resistance plasmid. The presence of the ColV plasmid or the combination of iss and tsh genes in the majority of APEC isolates supports the notion that when found together, the plasmid, iss, and tsh serve as good markers for APEC. These data indicate that different resistant E. coli pathotypes can be found in broiler chickens and that the distribution of such pathotypes and certain virulence determinants could be modulated by antimicrobial agent feed supplementation.

Hybridization on arrays was used to assess the presence of virulence-associated genes and to determine the relatedness of 32 non-O157 sorbitol-negative Escherichia coli isolates from healthy broiler chickens. These isolates were from commercial farms that used feed supplemented with different antimicrobial agents (virginiamycin, bacitracin, salinomycin, narasin, nicarbazin, or diclazuril). For each isolate, fluorescent probes were made from genomic DNA and were hybridized on DNA arrays composed of genes associated with general functions, virulence, iron uptake systems, and DNA repair genes (e.g., mut genes). Hybridization on arrays results showed that isolates from the same farm tended to be clustered but actually represented 18 genetically distinct groups of isolates. Results revealed that some isolates showed similarity to human uropathogenic E. coli or avian pathogenic E. coli. Four avian pathogenic E. coli-like isolates were detected. Another isolate possessed the intimin gene (eaeA) and typical genes of the type 3 secretion system associated with enteropathogenic E. coli and enterohemorrhagic E. coli strains. Genes from a second system (secondary type 3 secretion system) homologous to that found in Salmonella Typhimurium were detected in many isolates. Several of the studied isolates also possessed the aerobactin, salmochelin, and yersiniabactin genes involved in iron acquisition in pathogenic bacteria. Our results clearly suggest that commensal E. coli isolates from chickens are reservoirs of virulence-associated genes and may represent colibacillosis and zoonotic risks.

Food-producing animals can be reservoirs of pathogenic Escherichia coli strains that can induce diseases in animals or humans. Contamination of food by E. coli O157:H7 raises immediate concerns about public health, although it is not clear whether all E. coli O157 isolates of animal origin are equally harmful to humans. Inversely, the pathogenic potential of atypical E. coli O157 isolates and several non-O157 serotypes often is ignored. We used a DNA microarray capable of detecting a subset of 346 genes to compare the virulence-associated genes present in eight E. coli O157 isolates from human cases, 14 antibiotic-resistant and/or hypermutable E. coli O157 isolates from beef cattle, and four antibiotic-resistant, sorbitol-negative, non-O157 E. coli isolates from healthy broiler chickens. Hybridization on arrays (HOA) revealed that O157 isolates from beef cattle and humans were genetically distinct, although they possessed most of the same subset of virulence genes. HOA allowed discrimination between hypermutable and antibiotic-resistant O157 isolates from beef cattle based on hybridization results for the stx2 and ycgG genes (hypermutable) or ymfL, stx1, stx2, and hlyE(avian) genes (resistant). However, the absence of hybridization to gene yfdR characterized human isolates. HOA also revealed that an atypical sorbitol-fermenting bovine O157 isolate lacked some genes of the type 3 secretion system, plasmid pO157, and the stx1 and stx2 genes. This isolate had a particular pathotype (eaeA(beta) tir(alpha) espA(alpha) espB(alpha) espD(alpha)) not found in typical E. coli O157:H7. HOA revealed that some non-O157 E. coli isolates from healthy chickens carried genes responsible for salmochelin- and yersiniabactin-mediated iron uptake generally associated with pathogenic strains.

Antibacterial agents such as zinc bacitracin (ZB) and virginiamycin (VG) are used as growth promoting agents (GP) in broiler chicken production. The objective of this study was to evaluate the effect of the use of ZB and VG on the emergence of antibacterial resistance in a commercial broiler chicken farm. Three trials were conducted using 3 different diets: one without antibacterial agents, one containing VG, and one with ZB. Escherichia coli and Enterococcus spp. strains were isolated and tested for their susceptibility to various antibacterial agents. The occurrence of the resistance genes vatD, ermB, and bcrR in Enterococcus spp. isolates was determined by polymerase chain reaction (PCR). Comparative quantification of vatD and bcrR genes in total deoxyribonucleic acid (DNA) extracts from litter was done by SYBR Green Real-Time PCR (QPCR). Escherichia coli and Enterococcus spp. isolates from diet groups had different levels of resistance to various antibacterial agents over time. These GPs did not select for specific antibacterial agent resistance (AAR) in Enterococcus spp. The use of GPs seemed to lower the percentage of E. coli isolates resistant to some antibacterial agents. The presence of the bcrR gene could not explain all resistant phenotypes to ZB. Genes other than vatD and ermB might be involved in the resistance to VG in Enterococcus spp. Use of GPs was not associated with presence of the bcrR gene in DNA extracts from litter, but use of VG was associated with vatD presence.

The effects of feed supplementation with the approved antimicrobial agents bambermycin, penicillin, salinomycin, and bacitracin or a combination of salinomycin plus bacitracin were evaluated for the incidence and distribution of antibiotic resistance in 197 commensal Escherichia coli isolates from broiler chickens over 35 days. All isolates showed some degree of multiple antibiotic resistance. Resistance to tetracycline (68.5%), amoxicillin (61.4%), ceftiofur (51.3%), spectinomycin (47.2%), and sulfonamides (42%) was most frequent. The levels of resistance to streptomycin, chloramphenicol, and gentamicin were 33.5, 35.5, and 25.3%, respectively. The overall resistance levels decreased from day 7 to day 35 (P < 0.001). Comparing treatments, the levels of resistance to ceftiofur, spectinomycin, and gentamicin (except for resistance to bacitracin treatment) were significantly higher in isolates from chickens receiving feed supplemented with salinomycin than from the other feeds (P < 0.001). Using a DNA microarray analysis capable of detecting commonly found antimicrobial resistance genes, we characterized 104 tetracycline-resistant E. coli isolates from 7- to 28-day-old chickens fed different growth promoters. Results showed a decrease in the incidence of isolates harboring tet(B), bla(TEM), sulI, and aadA and class 1 integron from days 7 to 35 (P < 0.01). Of the 84 tetracycline-ceftiofur-resistant E. coli isolates, 76 (90.5%) were positive for bla(CMY-2). The proportions of isolates positive for sulI, aadA, and integron class 1 were significantly higher in salinomycin-treated chickens than in the control or other treatment groups (P < 0.05). These data demonstrate that multiantibiotic-resistant E. coli isolates can be found in broiler chickens regardless of the antimicrobial growth promoters used. However, the phenotype and the distribution of resistance determinants in E. coli can be modulated by feed supplementation with some of the antimicrobial agents used in broiler chicken production.

Antibiotic resistance patterns and the presence of antibiotic and virulence determinants in 74 sorbitol-negative Escherichia coli and 62 Salmonella isolates from nine different broiler chicken farms were investigated. Each farm was supplied by one of three companies that used different antimicrobial agents in feed for growth promotion. The isolates were identified by API 20E for E. coli and by serological tests for Salmonella. The susceptibility of the isolates to antibiotics was determined by Sensititre using the Clinical and Laboratory Standards Institute's breakpoints. Fifty-two E. coli isolates (70.3%) and nine Salmonella isolates (14.52%) were multiresistant to at least nine antibiotics. The multiresistant isolates were evaluated for the presence of tetracycline resistance, integron class 1, and blacMY 2 genes by PCR. Of the 74 E. coli isolates, 55 were resistant to amoxicillin and ceftiofur. Among these 55 resistant E. coli isolates, 45 (81.8%) and 22 (40.0%) were positive for blacMY-2 and qacEdeltal-Sull genes, respectively. Tetracycline resistance was found in 56 isolates (75.8%) among which 12 (21.4%) and 24 (42.9%) gave positive results for tetA and tetB, respectively. Virulence genes (iss, tsh, and traT), aerobactin operon (iucC), and the eaeA gene were detected in some E. coli strains. Among the 27 amoxicillin- and ceftiofur-resistant Salmonella isolates, the blacMY-2 gene was detected in 22 isolates. The class 1 integron gene (qacEdeltal-Sull) was not detected in any Salmonella isolates, whereas the invasin (inv) and virulence (spy) genes were found in 61 (98.4%) and 26 (42%) of the Salmonella isolates, respectively. This study indicated that multiple antibiotic-resistant commensal E. coli and Salmonella strains carrying virulence genes can be found on commercial broiler chicken farms and may provide a reservoir for these genes in chicken production facilities. Except for the presence of tetB, there was no significant effect of feed formulations on the phenotypic or genotypic characteristics of the isolates.