Antimicrobial Tolerance in Salmonella: Contributions to Survival and Persistence in Processing Environments.

Salmonella remains a top bacterial pathogen implicated in several food-borne outbreaks, despite the use of antimicrobials and sanitizers during production and processing. While these chemicals have been effective, Salmonella has shown the ability to survive and persist in poultry processing environments. This can be credited to its microbial ability to adapt and develop/acquire tolerance and/or resistance to different antimicrobial agents including oxidizers, acids (organic and inorganic), phenols, and surfactants. Moreover, there are several factors in processing environments that can limit the efficacy of these antimicrobials, thus allowing survival and persistence. This mini-review examines the antimicrobial activity of common disinfectants/sanitizers used in poultry processing environments and the ability of Salmonella to respond with innate or acquired tolerance and survive exposure to persists in such environments. Instead of relying on a single antimicrobial agent, the right combination of different disinfectants needs to be developed to target multiple pathways within Salmonella.

Changes in gene expression in the intestinal mucus of broilers with woody breast myopathy.

Previous work has shown that dietary treatments affect woody breast (WB) incidence differently, which indicates that gut conditions such as gut barrier function, inflammation, and oxidative stress are likely related to WB. In this study, dietary supplementation with antibiotics (bacitracin) or probiotics (Bacillus subtilis) was investigated for their effects on the expression of transcripts related to gut barrier function, inflammation, and oxidative stress in the mucus lining of the jejunum from broilers with or without WB. A split-plot experimental design was used in this study. The dietary treatments served as the main plot factor and the breast muscle condition was the subplot factor. On d 41, jejunum mucus was collected from 1 bird from each of 3 replicate pens in each 3 dietary treatment groups that exhibited WB and an additional bird that contained a normal breast (3 biological replicates/treatment/phenotype; 3 × 3 × 2, total N = 18). Total RNA was extracted using a commercial RNA extraction kit. The expression levels of CLDN1, MUC6, TLR2A, TLR2B, TLR4, IFN-γ, IL-1ß, IL-8L1, IL-10, NOS2, and SOD were determined using 2-step RT-qPCR analysis. The gene expression difference in ΔCt values was determined after normalizing with the chicken 18S rRNA gene. When the significant differences occurred between treatments, the relative fold change was calculated using the ΔΔCt method and the significance level was calculated. The PROC GLM procedure of SAS 9.4 was used, and the level of significance was set at P ≤ 0.05. There were no significant interactive effects between diet and the breast muscle condition on the expression of any of the genes tested. However, birds with WB exhibited higher MUC6 (P < 0.0001) gene expression levels than birds with normal breast muscles. In addition, the expression of SOD decreased in birds that were fed the antibiotic diet when compared to birds that were fed the probiotic diet (P = 0.014). In conclusion, WB identified in broilers tested in the current study is attributed to increased expression of mucin, indicating a correlation between WB incidence and gel-forming mucin secretion and pathogen signaling.

In silico prediction and expression analysis of vaccine candidate genes of Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is the most common food-borne pathogen that causes human gastroenteritis in the United States. Consumption of contaminated poultry products is considered as the major source of human Campylobacter infection. An effective vaccine would be a promising alternative to antibiotic supplements to curb C. jejuni colonization in poultry gastrointestinal (GI) tract. However, the genetic diversity among the C. jejuni isolates makes vaccine production more challenging. Despite many attempts, an effective Campylobacter vaccine is not yet available. This study aimed to identify suitable candidates to develop a subunit vaccine against C. jejuni, which could reduce colonization in the GI tract of the poultry. In the current study, 4 C. jejuni strains were isolated from retail chicken meat and poultry litter samples and their genomes were sequenced utilizing next-generation sequencing technology. The genomic sequences of C. jejuni strains were screened to identify potential antigens utilizing the reverse vaccinology approach. In silico genome analysis predicted 3 conserved potential vaccine candidates (phospholipase A [PldA], TonB dependent vitamin B12 transporter [BtuB], and cytolethal distending toxin subunit B [CdtB]) suitable for the development of a vaccine. Furthermore, the expression of predicted genes during host-pathogen interaction was analyzed by an infection study using an avian macrophage-like immortalized cell line (HD11). The HD11 was infected with C. jejuni strains, and the RT-qPCR assay was performed to determine the expression of the predicted genes. The expression difference was analyzed using ΔΔCt methods. The results indicate that all 3 predicted genes, PldA, BtuB, and CdtB, were upregulated in 4 tested C. jejuni strains irrespective of their sources of isolation. In conclusion, in silico prediction and gene expression analysis during host-pathogen interactions identified 3 potential vaccine candidates for C. jejuni.

Β-glucans and MOS, essential oil, and probiotics in diets of broilers challenged with Eimeria spp. and Clostridium perfringens.

The aim of this study was to evaluate the effectiveness of ß-glucans and MOS, essential oil (mixture of carvacrol and thymol), and the probiotic based on Saccharomyces boulardi, as an alternative to anticoccidial agents. In this experiment, six hundred seventy-two 1-day-old male broiler chicks were housed in batteries for 28 d. The experimental design of 4 randomized blocks with 24 cages each, and 7 birds per cage, consisted of an initial phase from 1 to 14 d of age, and a growth phase, from 15 to 28 d of age. The rations were formulated based on corn and soybean meal as energy and protein ingredients, respectively. All birds were inoculated with Eimeria spp and C. Perfringes at 14 d of age, and with C. Perfringes only at 21 d of age. The results showed the best weight gain in the initial phase when the anticoccidial agent was applied, whereas the use of additives in growth and entire experimental phases showed superior results in all treatments for this parameter. The birds that did not receive additives in the rations had the worst feed conversion in both phases and along raising. The results showed no significant differences between the treatments for the scores of lesions in the digestive tract and counts in the cecum, however, the red lesions increased numerically in the duodenum and jejunum of birds fed diets without the inclusion of additives. The use of additives indicated effectiveness for the parameters of performance in broilers challenged with C. Perfringes and Eimeria spp. at 14 d of age, and with C. Perfringes at 21 d of age.

The Prevalence of Salmonella and Campylobacter on Broiler Meat at Different Stages of Commercial Poultry Processing.

In poultry processing, Salmonella and Campylobacter contaminations are major food safety concerns. Peracetic acid (PAA) is an antimicrobial commonly used in commercial poultry processing to reduce pathogen prevalence so as to meet the USDA-FSIS performance standards. The objective of this study was to determine the prevalence of Salmonella and Campylobacter on broiler meat in various steps of commercial poultry processing in plants that use PAA. Post-pick, pre-chill, post-chill, and drumstick chicken samples were collected from three processing plants and mechanically deboned meat (MDM) was collected from two of the three plants. Each plant was sampled thrice, and 10 samples were collected from each processing step during each visit. Among the 420 samples, 79 were contaminated with Salmonella and 155 were contaminated with Campylobacter. Salmonella and Campylobacter contamination on the post-pick samples averaged 32.2%. Significant reductions in Salmonella and Campylobacter were observed in pre-chill to post-chill samples, where the prevalence was reduced from 34% and 64.4% to nondetectable limits and 1.1%, respectively (p < 0.001). Salmonella and Campylobacter remained undetectable on the drumstick samples in all three processing plants. However, the prevalence of Salmonella and Campylobacter on MDM was similar to the post-pick prevalence, which suggests substantial cross-contamination from post-chill to MDM.

Complete Genome Sequences of Four Campylobacter jejuni Strains Isolated from Retail Chicken Meat and Broiler Feces.

Campylobacter jejuni is the leading pathogen that causes foodborne infections. Here, we report the complete genome sequences of four C. jejuni strains isolated from retail chicken meat and broiler feces samples. Genes encoding type VI secretion and antibiotic resistance were detected among these isolates.

Internal organ and skeletal muscle development in commercial broilers with woody breast myopathy.

Increasing growth rate, body weight, and breast muscle yield have been linked to broiler muscle problems such as woody breast (WB). The aim of this study was to investigate the internal organ and skeletal muscle development of broilers with WB myopathy under dietary and Eimeria challenge treatments. A 3 diet (control, antibiotic, or probiotic) × 2 challenge (control or Eimeria) × 2 sex factorial arrangement of treatments was used in a randomized complete block design. Ross × Ross 708 chicks were randomly assigned to 96 floor pens with 12 treatment combinations (8 replicates per treatment). Internal organs were sampled on d 13 and 41. Skeletal muscles were sampled on d 41. Internal organ and skeletal muscle weights were analyzed using a 3-way analysis of variance (ANOVA). Relationships between WB and internal organ and skeletal muscle weights were analyzed using one-way ANOVA as all treatments were pooled together and regrouped according to WB scores. On d 41, absolute and relative heart weights were greater in males when they were averaged over diet and challenge treatments (P < 0.001 and P = 0.026, respectively). The birds with WB score 3 had greater absolute heart (P = 0.0002) and spleen weights (P = 0.016), but there was no difference in relative spleen weight (P > 0.05). When averaged over diet and challenge treatments, males have greater absolute duodenum, jejunum, and ileum weights (for all P < 0.0001). Compared with birds with normal breasts, the birds with WB scores 1, 2, and 3 had a greater live weight (for all P < 0.0001) and absolute and relative breast weights (for all P < 0.0001). The birds with WB score 1, 2, and 3 had greater (P < 0.0001) absolute but lower (P < 0.0001) relative drumstick, thigh, and wing weights. Results indicated that broilers with WB had lower relative proventriculus and gizzard weights and greater relative breast meat weight with lower relative drumstick, thigh, and wing muscle weights.

Spoilage Bacteria Counts on Broiler Meat at Different Stages of Commercial Poultry Processing Plants That Use Peracetic Acid.

In poultry processing, spoilage microbes are persistent microorganisms, which affect the quality of broiler meat. Peracetic acid (PAA) is the most common antimicrobial used by commercial processing plants, which can reduce the prevalence of these microbes. The goal of this study was to determine the concentrations of aerobic bacteria, coliforms, lactic acid bacteria, and Pseudomonas on broiler meat in processing plants that use peracetic acid in various concentrations as the primary antimicrobial. Samples were collected from three processing plants at five processing steps: post-pick (defeathering), pre-chill, post-chill, mechanically deboned meat (MDM), and drumsticks. Samples were rinsed in buffered peptone water for bacteria isolation. Over six log CFU/sample of aerobic plate counts (APC), lactic acid bacteria, and coliforms were detected on post-pick samples. All spoilage bacteria were reduced to nondetectable levels on post-chill samples (p < 0.001). However, the presence of all bacteria on mechanically deboned meat (MDM) samples indicated varying degrees of cross contamination from post-chill and MDM samples. These results suggest PAA effectively reduces spoilage microbes in chilling applications irrespective of differences in PAA concentrations. However, due to the levels of spoilage microbes detected in MDM, it may be worth investigating the potential interventions for this stage of processing.

Effects of dietary bacitracin or Bacillus subtilis on the woody breast myopathy-associated gut microbiome of Eimeria spp. challenged and unchallenged broilers.

Study suggested that dysbiosis of the gut microbiota may affect the etiology of woody breast (WB). In the current study, the cecal microbiota and WB in chickens fed three different diets were investigated. A total of 504 male chicks were used in a randomized complete block design with a 3 (Diet) × 2 (Challenge) factorial arrangement of treatments with 6 replicates per treatment, 6 treatments per block, and 14 birds per treatment. The experimental diets were a control diet (corn-soybean meal basal diet), an antibiotic diet (basal diet + 6.075 mg bacitracin/kg feed), and a probiotic diet (basal diet + 2.2 × 108 CFU Bacillus subtilis PB6/kg feed). On d 14, birds that were assigned to the challenge treatment received a 20 × live cocci vaccine. On d 41, breast muscle hardness in live birds was palpated and grouped into normal (NB) and WB phenotypes. Cecal contents were collected and their bacterial compositions were analyzed and compared. The genomic DNA of the cecal contents was extracted and the V3 and V4 regions of 16S rRNA gene were amplified and sequenced via an Illumina MiSeq platform. There were no differences (P > 0.05) in Shannon and Chao 1 indexes between the challenges, diets, and phenotypes (NB vs. WB). However, there was a difference (P = 0.001) in the beta diversity of the samples between the challenged and nonchallenged groups. Relative bacterial abundance differed (false discovery rate, FDR < 0.05) between the challenge treatments, but there were no significant differences (FDR > 0.05) among the three diets or two phenotypes. Predicted energy metabolism, nucleotide metabolism, and amino acid and coenzyme biosynthesis activities only differed (q-value < 0.05) between challenged and nonchallenged groups. The cocci challenge altered the gut microbial composition on Butyricicoccus pullicaecorum, Sporobacter termitidis, and Subdoligranulum variabile, but the dietary antibiotic and probiotic treatments did not impact gut microbial composition. No strong association was found between WB myopathy and gut microbial composition in this study.

Prevalence, Antimicrobial Resistance, and Molecular Characterization of Campylobacter Isolated from Broilers and Broiler Meat Raised without Antibiotics.

Campylobacter is one of the main bacterial pathogens that cause campylobacteriosis in the United States. Poultry is considered a major reservoir for the transmission of Campylobacter to humans. This study aimed to determine the prevalence and molecular characteristics of Campylobacter in the no-antibiotics-ever (NAE) broilers. A total of 414 samples were collected, among which 160 retail chicken samples were purchased from grocery stores and 254 samples were collected from broiler farms located in Mississippi State. The overall prevalence of Campylobacter was 25.4%, and a significantly higher prevalence was observed in retail chicken than in the farm samples (36.3% versus 18.5%; P < 0.0001), respectively. The prevalence of Campylobacter was not different (P = 0.263) between conventional retail (40.0%) and NAE (31.4%) retail chicken. Campylobacter jejuni was the predominant species among the positive isolates, accounting for 78.1%. Among the 82 C. jejuni isolates, 52.4% of the isolates carried the gyrA gene followed by the tet(O) gene (14.6%), whereas toxin-producing genes cdtA, cdtB, and cdtC were carried by 43.9%, 46.3%, and 43.9%, respectively. However, none of these virulence genes were detected in C. jejuni isolated from litter samples. Among tested C. jejuni, 13.6% of the isolates were multidrug resistant. The highest resistance was observed against nalidixic acid (49.2%), followed by tetracycline (23.7%). Our study suggests that the prevalence of Campylobacter was higher in retail meat samples than in environmental samples obtained from farms, and there was no difference in Campylobacter prevalence among conventional and NAE retail chicken. IMPORTANCE The FDA antibiotic withdrawal policy has led to a shift in the production system, from conventional antibiotics fed birds to no antibiotics ever (NAE) raised birds. However, the impact of this shift to NAE on the prevalence and characteristics of Campylobacter has not been studied on the farm or in retail chicken meats. The objective of this study was to determine the current prevalence of Campylobacter and the distribution of their antimicrobial resistance and virulence genes in NAE-raised broilers. The findings of this study will help the industry to take necessary action to develop effective mitigation strategies for reducing Campylobacter contamination in NAE broilers.

Bacitracin, Bacillus subtilis, and Eimeria spp. challenge exacerbates woody breast incidence and severity in broilers.

Woody breast (WB) is a myopathy that is related to the increasing growth rate. Understanding the influence of management factors on WB formation and development is important to minimize WB. This study was conducted to define how management factors affect broiler growth performance, processing yield, and WB incidence. Ross × Ross 708 chicks were randomly assigned to a 3 (diet) × 2 (cocci challenge) × 2 (sex) factorial arrangement of treatments. The 3 dietary treatments were: control diet (corn-soybean meal basal diet), antibiotic diet (basal diet + 6.075 mg bacitracin /kg feed), and probiotic diet (basal diet + 2.2 × 108 CFU Bacillus subtilis PB6/kg feed). Birds in cocci challenge treatments received 20 × live cocci vaccine on d 14. The hardness of breast muscle in live birds was determined by palpation and grouped into Normal, Slight, Moderate, and Severe categories. Across diet and sex treatments, the cocci challenge resulted in decreases in body weight (BW) on d 29 and 35 (P < 0.0001 and = 0.032) in body weight gain (BWG) from d 14 to 29 (P < 0.0001). However, an increase of BW occurred on d 35 (P = 0.032) and an increase of BWG occurred from d 29 to 35 and d 35 to 43 (P = 0.0001 and 0.002), and the cocci challenge increased WB incidence on d 29 (P = 0.043) and d 43 (P = 0.013). Across challenge and sex treatments, birds fed the antibiotic diet exhibited a higher growth rate (GR) than those fed the control or probiotic diet from d 0 to 14 (P = 0.016), but not after d 14 (P > 0.05). Across sex, the antibiotic and probiotic diets increased WB incidence for those birds that did not receive a cocci challenge on d 43 (P = 0.040). Across challenge and diet treatments, males exhibited a higher BW, BWG, and GR throughout all growth phases, and males showed a higher WB incidence on d 29, 35, and 43 (P = 0.002, P < 0.0001, and P = 0.0002, respectively). In conclusion, bacitracin and Eimeria spp. increased WB incidence, BW, and GR. However, Bacillus subtilis increased WB incidence in male broilers without affecting BW and GR.

Effect of housing environment and hen strain on egg production and egg quality as well as cloacal and eggshell microbiology in laying hens.

This study was conducted to determine the effect of housing environment and laying hen strain on performance, egg quality, and microbiology of the cloaca and eggshell. A total of 1,152 Hy-Line Brown (HB) and Hy-Line W-36 White Leghorn (W-36) hens were used. All hens were kept in conventional cages (CC) from 18 to 32 wk of age and then moved to either enriched colony cages (EC) or free-range (FR) pens or continued in CC. Hens were randomly allocated into a 2 × 3 factorial arrangement of 2 laying hen strains (brown and white) and 3 housing environments (CC, EC, and FR) in a split plot in time (hen age) design. The experiment was conducted from 32 to 85 wk of age. The experiment was divided into 2 phases: early phase (32-51 wk of age) and late phase (52-85 wk of age). A 3-way interaction was observed for hen day egg production (HDEP) among housing environments, hen strain, and bird age in the early phase (P = 0.004) as well as in the late phase (P < 0.0001). In both of the phases, HDEP was higher in CC and FR than in EC. Hy-Line W-36 hens raised in EC had the lowest HDEP compared to other treatments. A 3-way interaction was observed for feed intake (FI; P = 0.017) and feed conversion ratio (FCR) in the late phase (P < 0.0001). The lowest FI and highest FCR were observed in EC for W-36 hens. Free-range hens performed in-between for eggshell quality when compared to CC and EC while HB had better egg quality than W-36. Free-range hens had higher cloacal bacterial counts for aerobes, anaerobes, and coliforms than CC and EC. Higher eggshell bacterial contamination was observed in eggs from FR versus eggs from CC and EC. These results indicate that both housing environment and laying hen strain affect performance and egg quality as well as cloacal and eggshell microbiology. Further studies should be conducted to determine food safety and economic impacts when using different hen strains and housing environments.

In ovo administration of Bacillus subtilis serotypes effect hatchability, 21-day performance, and intestinal microflora.

Recent research has tried to maximize broiler chick health and performance by utilizing commercial in-feed probiotics to inoculate fertile hatching eggs, and thus expose birds earlier to beneficial bacteria. However, the in ovo inoculation of a specific serotype of Bacillus subtilis was detrimental for broiler hatchability. Therefore, the objective of this study was to determine if other B. subtilis serotypes negatively affect hatchability or if it is associated with a specific serotype. It was also of interest to determine if the B. subtilis serotype influence chick performance and intestinal microflora. On d18 of incubation, 1886 fertile broiler eggs were in ovo inoculated with the following treatments (T): T1 = Marek's vaccine (MV), T2 = MV + B. subtilis (ATCC 6051), T3 = MV + B. subtilis (ATCC 8473), and T4 = MV + B. subtilis (ATCC 9466). It should be noted that in a previous study, T2 was detrimental to hatchability. Inoculated eggs were transferred to 3 hatchers/T. At hatch, chicks were weighed, feather sexed, and hatch residue analysis was conducted. Male chicks were randomly assigned to 40 raised wire cage so that there were 10 birds/cage. On d 0, 7, 14, and 21 of the grow-out, chicks and feed were weighed to calculate performance data. On these days, the ileum and ceca were aseptically collected to enumerate total aerobes and coliforms. No differences were observed for percentage of mid dead embryos, cracked eggs, and cull chicks (P > 0.05). However, hatch of transfer was significantly reduced by T2 compared to T1, T3, and T4 (P < 0.001). T2 had significantly higher percentages of late dead embryos and pips when compared to the other treatments (P = 0.002 and P < 0.001, respectively). Chicks hatched from T2 were not vigorous and, thus, not used for the grow-out trial. No differences were observed for growth performance characteristics for any of the treatments (P > 0.05). For bacterial enumeration, the ileum had equal or fewer bacterial counts for T3 and T4 when compared to T1 on most sampling days, except on d21 where T4 had higher aerobic and coliform counts (P ≤ 0.0001). For the ceca, T3 and T4 had equal or fewer bacterial counts than T1 on every sampling day (P ≤ 0.0001). These data demonstrate that not all B. subtilis evaluated are detrimental to hatchability, but rather, serotype dependent. In addition, different B. subtilis serotypes can modify the intestinal microflora with potential to reduce pathogenic bacteria present in young broiler, without impacting overall performance.

Effects of in ovo probiotic administration on the incidence of avian pathogenic Escherichia coli in broilers and an evaluation on its virulence and antimicrobial resistance properties.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, which has been traditionally controlled by the prophylactic in-feed supplementation of antibiotics. However, antibiotics are being removed from poultry diets owing to the emergence of multidrug-resistant (MDR) bacteria. Therefore, alternatives to control APEC are required. This study aimed to evaluate the effects of in ovo inoculation of probiotics on the incidence of APEC in broilers and evaluate the virulence and antimicrobial resistance properties of the APEC isolates. On embryonic day 18, 4 in ovo treatments (T) were applied: T1 (Marek's vaccine [MV]), T2 (MV and Lactobacillus animalis), T3 (MV and Lactobacillus reuteri), and T4 (MV and Lactobacillus rhamnosus). A total of 180 male broilers per treatment were randomly placed in 10 pens. The heart, liver, spleen, and yolk sac were collected on day 0, 14, 28, and 42. Presumptive E. coli isolates were confirmed by real-time PCR. The positive isolates were screened for the APEC-related genes (iroN, ompT, hlyF, iss, and iutA), and E. coli isolates containing one or more of these genes were identified as APEC-like strains. A total of 144 APEC-like isolates were isolated from 548 organ samples. No differences (P > 0.05) among treatments were observed for the incidence of APEC-like strains in all organs when averaged over sampling days. However, when averaged over treatments, the incidence in the heart, liver, and yolk sac was different among sampling days; a significant increase was observed in these organs on day 14 compared with day 0. Twenty-five antimicrobial resistance genes were evaluated for all APEC-like isolates, and 92.4% of the isolates carried at least one antimicrobial resistance gene. Thirty-seven isolates were then selected for antimicrobial susceptibility testing; MDR strains accounted for 37.8% of the isolates. In conclusion, the in ovo inoculation of a single probiotic strain did not confer protection against APEC strains in broilers. The high prevalence of MDR isolates indicates that further research on antibiotic alternatives is required to prevent APEC infections in broilers.

Complete genome sequence of multidrug-resistant avian pathogenic Escherichia coli strain APEC-O2-MS1170.

OBJECTIVES: Avian pathogenic Escherichia coli (APEC) causes colibacillosis, one of the leading causes of mortality and morbidity associated with significant economic losses in the poultry industry. This study aimed to determine antimicrobial resistance and to characterise the genome sequence of a multidrug-resistant (MDR) APEC strain isolated from a broiler chicken. METHODS: Strain APEC-O2-MS1170 was isolated from the broiler yolk sac of a 14-day-old broiler. Antimicrobial susceptibility testing was performed using a Sensititre National Antimicrobial Resistance Monitoring System (NARMS) Gram-negative panel. Whole-genome sequencing was performed using both the long-read sequencing approach with a Nanopore GridION sequencer and short-read sequencing with an Illumina HiSeq X-Ten sequencer to obtain a complete scaffold of the genome and an accurate sequence. RESULTS: The genome size of strain APEC-O2-MS1170 is 4,993,909 bp with a GC content of 50.7% and 4,651 protein-coding sequences. Public databases were used to identify the virulence-associated gene and antimicrobial resistance gene cargo. Plasmid comparison showed that pAPEC-O2-MS1170-R is a large multidrug resistance IncB/O/K/Z plasmid, while pAPEC-O2-MS1170-ColV shares homology with the APEC ColV virulence plasmid. CONCLUSION: The genome sequence of APEC-O2-MS1170 provides valuable information on resistance mechanisms and virulence characteristics of pathogenic E. coli as well as information for tracing the potential spread of this MDR strain.

In ovo inoculation of an Enterococcus faecium-based product to enhance broiler hatchability, live performance, and intestinal morphology.

Previous studies have suggested the use of probiotics, as alternative to antibiotics, to enhance broiler performance. The administration of probiotics in feed has been widely explored; however, few studies have evaluated the in ovo inoculation of probiotics. Therefore, the objective was to evaluate the impact of in ovo inoculation of different concentrations of GalliPro Hatch (GH), an Enterococcus faecium-based probiotic, on hatchability, live performance, and gastrointestinal parameters. Ross x Ross 708 fertile eggs were incubated, and on day 18, injected with the following treatments: 1) 50 µL of Marek's vaccine (MV), 2) MV and 1.4 × 105 cfu GH/50 µL, 3) MV and 1.4 × 106 cfu GH/50 µL, 4) MV and 1.4 × 107 cfu GH/50 µL. On the day of hatch, chicks were weighed, feather sexed, and hatch residue was analyzed. Male birds (640) were randomly assigned to 40 floor pens. On day 0, 7, 14, and 21 of the grow-out phase, performance data were collected. One bird from each pen was used to obtain yolk weight and intestinal segment weight and length. Hatchability was not impacted by any GH treatment (P = 0.58). On day 0, yolk weight was lower for all treatments than for MV alone. On day 0 to 7, feed intake was lower for 105 and 107 GH; the feed conversion ratio (FCR) was lower for all treatments than for MV alone (P = 0.05; P = 0.01, respectively). From day 14 to 21, the 107 GH treatment had higher BW gain (P = 0.05). For day 0 to 21, 107 GH had a lower FCR than MV alone (P = 0.03). On day 0, all GH treatments resulted in heavier tissues and longer jejunum, ileum, and ceca lengths than MV alone (P < 0.05). Spleen weight was higher for 105 and 107 GH than for MV alone. In conclusion, GH does not impact hatchability, and some concentrations improved live performance through the first 21 d of the grow-out phase. These improvements could result from the increased yolk absorption and improved intestinal and spleen morphology seen in this study.

Avian Pathogenic Escherichia coli and Clostridium perfringens: Challenges in No Antibiotics Ever Broiler Production and Potential Solutions.

United States is the largest producer and the second largest exporter of broiler meat in the world. In the US, broiler production is largely converting to antibiotic-free programs which has caused an increase in morbidity and mortality within broiler farms. Escherichia coli and Clostridium perfringens are two important pathogenic bacteria readily found in the broiler environment and result in annual billion-dollar losses from colibacillosis, gangrenous dermatitis, and necrotic enteritis. The broiler industry is in search of non-antibiotic alternatives including novel vaccines, prebiotics, probiotics, and housing management strategies to mitigate production losses due to these diseases. This review provides an overview of the broiler industry and antibiotic free production, current challenges, and emerging research on antibiotic alternatives to reduce pathogenic microbial presence and improve bird health.

Effects of Housing Types on Cecal Microbiota of Two Different Strains of Laying Hens During the Late Production Phase.

Due to animal welfare issues, European Union has banned the use of conventional cages (CC) and non-EU countries including the US are also under constant public pressure to restrict their use in egg production. Very limited information is available on the composition of the microbial community of hens raised in different housing environments. This study was conducted to determine the effects of CC and enriched colony cages (EC) on cecal microbiota of two commercial laying hen strains, Hy-Line W36 (W36) and Hy-Line Brown (HB) during the late production stage (53, 58, 67, and 72 weeks of age). Cecal microbiota was studied by analyzing 16S rRNA gene sequences with Quantitative Insights Into Microbial Ecology (QIIME) 2 ver. 2018.8. Differentially abundant taxa were identified by Linear discriminant analysis Effect Size (LEfSe) analysis (P < 0.05, LDA score > 2.0). At phylum level, Actinobacteria was significantly enriched in W36 at all time points while Synergistetes (53 weeks), Spirochaetes (58 weeks), and Synergistetes and Spirochaetes (67 weeks) were significantly higher in HB. At genus level, Bifidobacterium (at all time points) and butyric acid producing genera such as Butyricicoccus and Subdoligranulum (58 and 72 weeks) were significantly higher in W36 as compared to HB. Moreover, Proteobacteria (72 weeks) and its associated genus Campylobacter (67 and 72 weeks) were significantly enriched in EC as compared to CC. Alpha diversity was significantly higher in HB (at all time points) and in EC (67 weeks) as compared to W36 and CC, respectively. Similarly, there was a significant difference in community structure (beta diversity) between W36 and HB (all time points) as well as between EC and CC (67 weeks). The effect of housing and strains was not only seen at the bacterial composition and structure but also reflected at their functional level. Notably, KEGG metabolic pathways predicted to be involved in carbohydrates degradation and amino acids biosynthesis by PICRUSt analysis were significantly different between W36 and HB housed at CC and EC. In sum, cecal microbiota composition, diversities, and their functional pathways were affected by housing type which further varied between two commercial laying hen strains, HB and W36. This suggests that both housing and genetic strains of laying hens should be considered for selection of the alternative housing systems such as enriched colony cage.

The potential for inoculating Lactobacillus animalis and Enterococcus faecium alone or in combination using commercial in ovo technology without negatively impacting hatch and post-hatch performance.

The poultry industry has recently undergone transitions into antibiotic free production, and viable antibiotic alternatives, such as probiotics, are necessary. Through in ovo probiotic inoculation, beneficial microflora development in the gastrointestinal tract may occur prior to hatch without negatively impacting chick performance. Therefore, the objective of the present study was to observe the impacts of the injection of probiotic bacteria individually or combined into fertile broiler hatching eggs on hatch and live performance characteristics. A total of 2,080 fertile broiler hatching eggs were obtained from a commercial source. On day 18 of incubation, 4 in ovo injected treatments were applied: 1.) Marek's Disease (HVT) vaccination, 2.) L. animalis (∼106 cfu/50µl), 3.) E. faecium (∼106 cfu/50µl), and 4.) L. animalis + E. faecium (∼106 cfu & ∼106 cfu/50µl each). On day of hatch, hatchability and hatch residue data were recorded. A portion of male chicks from each treatment were placed in a grow-out facility for a 21 d grow-out (18 chicks/pen × 10 pens/treatment = 720 male chicks) with a corn and soy bean meal-based diet without antibiotics or antibiotic alternatives. Performance data and gastrointestinal samples were collected on days 0, 7, 14, and 21. Results indicated no differences in all hatch parameters between treatments (P > 0.05) except for % pipped, where the L. animalis treatment had lower % pipped eggs compared to the HVT control and E. faecium treatments (P = 0.04). No differences were observed in body weight gain or mortality (P > 0.05). Probiotic treatments altered gastrointestinal tissue length, weight, and pH. This resulted in all in ovo injected probiotic treatments increasing feed conversion ratio (FCR) from days 7 to 14 as compared to the control (P = 0.01). Differences in FCR were not observed in any other week of data collection (days 0 to 7, 14 to 21, or 0 to 21; P > 0.05). Although probiotics altered live performance from days 7 to 14, these data suggest that in ovo inoculations of L. animalis and E. faecium in combination are viable probiotic administration practices that potentially improve hatch characteristics and gastrointestinal tract development.

Post hatch recovery of a probiotic Enterococcus faecium strain in the yolk sac and intestinal tract of broiler chickens after in ovo injection.

Concerns about antibiotic-resistant bacteria and their presence in animal products grow and thus alternatives to use of antibiotics in animal production are being investigated. Probiotics have gained increased focus due to improvements in performance, immune health and pathogen reduction when provided to poultry through feed. These traits may be further improved if probiotics can be provided to the embryo before hatch, before meeting environmental pathogens. The objective was to determine the faith of a probiotic Enterococcus faecium (M74) strain in the yolk sac and intestinal tract of broiler chickens after injection into hatching eggs. E. faecium M74 (1.4 × 107 CFU/egg) was applied in ovo at day 18 of incubation. From 1- and 7-day-old chickens, 20 samples from yolk sac, caecal tonsils and rest of the intestinal tract were subjected to CFU counting. Isolates from a sample subset were typed by pulsed-field gel electrophoresis (PFGE). Enterococci were found in varying numbers: 1.0 × 104-2.2 × 1010 CFU/g. The prevalence of M74 PFGE profiles was high in 1-day-old (88%) and 7-day-old chickens (67%). This demonstrates that the embryos ingested M74 before hatching, that M74 is viable for intestinal colonization through in ovo administration, and that the strain multiplies in the chickens gastrointestinal tract post hatching.