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Commercial hatch cabinet environments promote replication of microorganisms. These pathogenic or apathogenic microorganisms may serve as pioneer colonizers of the gastrointestinal tract (GIT) of poultry. Some of these pioneer colonizers, such as Escherichia coli and Enterococcus spp., are opportunistic pathogens that lead to reduced performance in commercial poultry. Effective hatchery sanitation is imperative to limit contamination of naïve neonatal chicks and poults. Formaldehyde fumigation has been traditionally used to reduce the pathogen load in commercial hatch cabinets. To investigate potential alternatives to formaldehyde fumigation, models to mimic the microbial bloom in a laboratory setting must be utilized. The purpose of the present study was to evaluate the impact of a multispecies environmental challenge model (PM challenge) with and without formaldehyde fumigation during the hatching phase on early performance in broiler chicks. Three experiments were conducted to evaluate microbial contamination in the hatch cabinet environment (air samples, fluff samples), enteric colonization at day-of-hatch (DOH), and 7-day performance. In all experiments, significantly (P < 0.05) more gram-negative bacteria were recovered from the GIT at DOH in the PM challenge control group as compared to the nonchallenged control (NC) group and the formaldehyde-treated group (PM + F). There were no statistical differences in 7-day body weight gain or feed conversion ratio between the PM challenge control group, the NC group or the PM + F group. These data suggest this model could be utilized to evaluate alternatives to formaldehyde fumigation for controlling the microbial load during the hatching phase in a laboratory setting., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The objective of this experiment was to investigate the effects of the dietary soy galactooligosaccharides (GOS), raffinose and stachyose, on performance, gastrointestinal health, and systemic stress in young broilers. Birds were fed a GOS-devoid diet based on soy protein isolate (SPI) or the SPI diet with 0.9, 1.8, 2.7, or 3.6% added stachyose and raffinose in a ratio of 4:1 at the expense of corn starch. These 5 treatments were administered to 10 replicate cages of 8 birds. Performance was measured weekly and excreta moisture, N retention, apparent metabolizeable energy, and complete blood cell counts were determined at 14 and 21 d. At 21 d, 2 birds per cage were orally gavaged with fluorescein isothiocyanate-dextran (FITC-d) and serum samples were analyzed for FITC-d as a marker of gut leakage. Additionally, intestinal morphology, crop presumptive lactic acid bacteria (LAB) counts, crop and cecal pH, and cecal microbiota via16S rRNA microbial sequencing were evaluated at 21 d. From 0 to 21 d, feed intake increased linearly (P < 0.01) as dietary GOS increased, whereas BWG increased (P < 0.05) quadratically. Feed conversion ratio increased (P < 0.01) linearly as GOS increased. There were linear increases (P < 0.05) in excreta moisture as dietary GOS increased at 14 and 21 d, as well as dose-dependent responses (P < 0.05) in N retention, AME, and AME n . There was a quadratic increase (P < 0.05) in crop LAB recovery and a linear decrease (P < 0.01) in ceca pH as GOS increased. At 14 d, a linear increase (P < 0.05) in blood heterophil to lymphocyte ratio was observed as dietary GOS increased. Serum concentrations of FITC-d increased quadratically (P < 0.01) to dietary GOS. Increasing levels of GOS influenced alpha and beta diversities and composition of gut microbiota, including the abundance of Ruminococcus and Bifidobacterium. Results from this trial indicate that soy-derived GOS exert dose-dependent effects on nutrient utilization and intestinal health in young broilers., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Microbial blooms that emerge in commercial hatch cabinets consist of apathogenic and pathogenic microorganisms, including Escherichia coli, Enterococcus faecalis, and Aspergillus fumigatus. Objectives of the present study included the development of a multipathogen contamination model to mimic commercial conditions and optimization of sampling methods to quantify bacterial or fungal presence within the hatch cabinet. The pathogen challenge mix (PM) was recreated from select bacterial or fungal isolates recovered from an egg homogenate (EH) derived from the contents of infertile eggs and late embryonic mortalities. Isolates selected for PM included Enterococcus faecalis (∼10 8 CFU/egg), Staphylococcus aureus (∼10 7 CFU/egg), Staphylococcus chromogenes (∼10 7 CFU/egg), Aspergillus fumigatus (∼10 6 spores/egg), and 2 Escherichia coli (∼10 8 CFU/egg) isolates. Challenge (100 μL of PM or EH) was administered using a sterile loop to a 28 mm area on the blunt end of the eggshell at day 19 of embryogenesis (DOE). In 3 experiments, microbiological data were collected from environmental hatcher samples (open-agar plate method), fluff samples, postmortem whole-body chick rinse samples, and gastrointestinal tract (GIT) samples to evaluate select bacteria and fungi circulating within the hatch cabinet and colonization of GIT. Cumulative bacterial and fungal recovery from the PM hatching environment from DOE20 to hatch was higher than the nonchallenged group (NC) and EH group at ∼860 and ∼1,730 CFU, respectively. Bacterial recovery from GIT, fluff, and chick rinse samples were similar for the PM and EH group in Exp. 1. However, Aspergillus fumigatus recovery from fluff and chick rinse samples for the PM group was significantly (P < 0.001) higher than the NC and EH group. In Exp. 2 and 3, PM challenge significantly (P < 0.05) increased Gram-negative bacterial recovery from the GIT, fluff and chick rinse samples compared to both the NC and EH group. These data suggest this innovative multispecies environmental contamination model using PM could be utilized to evaluate strategies to mitigate microbial contamination in commercial hatch cabinets in a laboratory setting., Competing Interests: DISCLOSURES The authors have no conflicts of interest to report., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Avian pathogenic Escherichia coli (E. coli) is an opportunistic pathogen often introduced to neonatal chicks during the hatching process. This commensal bacterium, particularly as a pioneer colonizer of the gastrointestinal tract, can have substantial implications in the rearing of poultry because of reduced flock performance. In order to mimic the effects of the natural bacterial bloom present during the hatch, a seeder challenge model was developed to expose neonatal chicks to virulent E. coli. On day 20 of embryogenesis, selected early hatched chicks (n = 18/hatcher) were briefly removed and sprayed challenged with saline (vehicle) or E. coli at 1 × 10 7  colony-forming unit (CFU)/chick (exp 1) and 2.5 × 10 7  CFU/chick (exp 2). These challenged chicks were returned to the hatcher to serve as seeders to transmit the pathogen to the indirect challenged or contact chicks (n = 195/hatcher). For two 7-d experiments, the efficacy of transmission was evaluated via enteric bacterial recovery, body weight gain (BWG), and mortality. For exp 1 and exp 2, significantly (P < 0.0001) more gram-negative bacteria were recovered from the seeder and contact gastrointestinal samples than the negative control samples on day of hatch. In addition, there was a reduction (P < 0.05) in 7-d BWG and significantly (P < 0.0001) higher mortality in the contact-challenged chicks than the negative control chicks in both exp 1 and exp 2. These data suggest that this challenge model could be used to evaluate different methods of controlling the bacterial bloom that occurs in the hatching environment., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Horizontal transmission of opportunistic Escherichia coli during hatch can have detrimental effects on early performance, particularly as pioneer colonizers. Commercially, formaldehyde is often applied in the United States to combat the bacterial bloom that occurs inside of the hatching environment. The purpose of these experiments was to develop a replicable E. coli horizontal challenge model to evaluate alternatives to formaldehyde sanitation applied to the hatching environment. In experiment 1, two trials were conducted for 2 wild-type (WT) E. coli isolates (isolate 1 [I1] or isolate 2 [I2]) to determine the appropriate in ovo challenge dose and day of embryogenesis (DOE) for challenge administration. In experiment 1 trial 1, the most appropriate inoculation dose and time point were determined to be 10 2  cfu/embryo on DOE 19. Experiment 1 trial 2 evaluated whether placement of seeder (direct-challenged) embryos with contact (indirect-challenged) embryos during hatch affected contact hatchability. Trial 2 showed no differences in hatchability between groups. A 7-day experiment (experiment 1 trial 2) was conducted to evaluate the effects of I1 or I2 on horizontal transmission, gram-negative bacterial (GNB) recovery from the gastrointestinal tract (GIT), and impact on BW gain (BWG). Compared with the negative control, seeder, and contact chicks challenged with I1 or I2, we observed increased (P < 0.05) GNB recovered from GIT on the day of hatch. There was a marked (P < 0.05) reduction in 7-day BWG between the I1 indirect-challenged group and the negative control group. To further validate the model, 2 7-day trials (experiment 2, experiment 3) were conducted to evaluate the effects of formaldehyde fumigation on coliform recovery from the hatching environment and on early performance using I1 for the challenge. Isolate 1 positive control hatchers had increased levels of circulating coliforms compared with the negative control and formaldehyde-treated hatchers, although there was no significant impact on performance induced by challenge or formaldehyde treatment in experiment 2 or experiment 3. These data provide a potential model for investigations related to horizontal transmission of WT E. coli at a low dose on DOE 19 to promote simulated commercially relevant bacterial blooms under laboratory conditions., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

This study's objective was to evaluate the effect of the fermented soybean meal (FSBM) on Salmonella typhimurium (ST) to turkey poults using two models of infection. In the prophylactic model, one-day-old turkeys were randomly allocated to one of four different groups ( n = 30 turkeys/group): (1) Control group, (2) FSBM group, (3) Control group challenged with ST (Control + ST), and (4) FSBM group challenged with ST (FSBM + ST). On day 9 of age, all poults were orally challenged with 10 6 colony forming units (CFU) ST and 24 h post-inoculation, intestinal samples were collected to determine ST recovery and morphometric analysis. Blood samples were collected to evaluate serum fluorescein isothiocyanate-dextran (FITC-d). In the therapeutic model, a similar experimental design was used, but turkeys were orally gavaged 10 4 CFU ST on day 1, and samples were collected at day 7. FSBM improved performance and reduced leaky gut in both experimental infective models. In the prophylactic model, FSBB induced morphology changes in the mucosa. Although the strains ( Lactobacillus salivarius and Bacillus licheniformis ) used for the fermentation process showed in vitro activity against ST, no significant effect was observed in vivo. The fermentation with different beneficial bacteria and different inclusion rates of FSBM requires further investigation.

During the hatching process, chicks are exposed to opportunistic and/or pathogenic organisms, such as virulent or avirulent Escherichia coli. Virulent E. coli strains have not been feasible for induction of neonatal colibacillosis via in ovo challenge due to high embryonic mortality. In this manuscript, we describe the addition and co-administration of the bacteriostatic antibiotic tetracycline to a virulent E. coli challenge culture, improving hatchability and livability of seeder chicks while allowing robust horizontal transmission in the hatching cabinet to contact chicks. Experiment 1 consisted of 3 trials. Experiment 1, trial 1 was conducted to determine an effective ratio of E. coli challenge and tetracycline dose to be utilized in the seeder model. Trials 2 and 3 were conducted to evaluate the transmission of E. coli from seeder to contact chicks. Experiment 2 consisted of 3 independent 7-D trials where body weight gain (BWG), mortality, and selected enteric bacterial recovery were evaluated. In trials 1 to 3, significantly (P < 0.05) more Gram-negative bacteria were recovered from whole gut samples (GIT) vs. negative controls on day of hatch, from both seeder and contact chicks. At day 7 in trial 1, contact chicks had significantly (P < 0.05) more Gram-negative bacteria recovered from the GIT than the negative control, but not in trials 2 and 3. Presumptive lactic acid bacterial recovery was elevated in contact and seeder chicks compared to the negative control in all 3 trials. Contact challenge caused a significant (P < 0.05) reduction in BWG in 2 out of 3 trials at day 7, and there was a significant (P < 0.05) increase in mortality as compared to the negative controls in all trials. These data suggest that co-administration of a virulent E. coli strain with tetracycline allows for hatch of direct challenged chicks and effective horizontal transmission to contact chicks during the hatching process, as evidenced by reduced day 7 performance and altered selected enteric bacterial recovery., (© 2019 Poultry Science Association Inc.)

Necrotic enteritis (NE) is a recognized multifactorial disease that cost annually to the poultry industry around $2 billion. However, diverse aspects related to its presentation are not completely understood, requiring further studies using known induction experimental models. Therefore, the purpose of this study was to measure the changes occurring in performance, intestinal integrity and ileal microbiome using a previously established NE-challenge model. Chickens were assigned to a negative control group (NC) or a positive control group (PC). In the PC, broilers were orally gavaged with Salmonella Typhimurium (ST) (1 × 10 7 cfu/chick) at day 1, Eimeria maxima (EM) (2.5 × 10 4 oocyst/chick) at day 18 and Clostridium perfringens (CP) (1 × 10 8 cfu/chick/day) at 23-24 days of age. Weekly, body weight (BW), body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) were evaluated. Morbidity and mortality were determined throughout the study, and NE lesion scores were recorded at day 25. Additionally, blood and liver samples were collected to measure gut permeability as determined by levels of serum fluorescein isothiocyanate-dextran (FITC-d) and bacterial translocation (BT). Ileal contents were processed for 16S rRNA gene-based microbiome analysis. Performance parameters and intestinal permeability measurements were negatively impacted in the PC resulting in elevated serum FITC-d and BT with a -6.4% difference in BWG. The NE lesion score in PC (1.97 vs. 0.00) was significantly higher in comparison to NC, although there was no difference in mortality. The microbiome analysis showed a dramatic shift of ileal microbiomes in PC groups as compared to NC (ANOSIM: R = 0.76, P = 0.001). The shift was characterized by reduced abundance of the phylum Actinobacteria ( P < 0.01), and increased abundance of the genera Butyrivibrio, Lactobacillus, Prevotella and Ruminococcus in PC compared to NC ( P < 0.05). Expectedly, Clostridium was found higher in PC (2.98 ± 0.71%) as compared to NC (1.84 ± 0.36%), yet the difference was not significant. In conclusion, results of the present study showed the different intestinal epithelial and microbiological alterations occurring in an established NE-challenge model that considers paratyphoid Salmonella infections in young chicks as an important predisposing factor for presentation of NE.

The present study evaluated the microbiological properties of three probiotic candidate strains of lactic acid bacteria (LAB) (128; 131; CE11&#95;2), their effect on intestinal epithelial permeability, and their ability to reduce intestinal colonization of Salmonella Typhimurium (ST) individually or as a batch culture in neonatal turkey poults. Isolates were characterized morphologically and identified using 16S rRNA sequence analyses. Each isolate was evaluated for tolerance and resistance to acidic pH, high osmotic NaCl concentrations, and bile salts in broth medium. In vitro assessment of antimicrobial activity against different enteropathogenic bacteria was determined using an overlay technique. In vitro intestinal permeability was evaluated using a stressed Caco-2 cell culture assay treated with/without the probiotic candidates. The in vivo effect of the selected LAB strains on ST cecal colonization was determined in two independent trials with neonatal turkey poults. The results obtained in this study demonstrate the tolerance of LAB candidates to pH 3, a NaCl concentration of 6.5%, and high bile salts (0.6%). All strains evaluated exhibited in vitro antibacterial activity against Salmonella Enteritidis, ST, and Campylobacter jejuni. Candidates 128 and 131 exhibited a coccus morphology and were identified as Enterococcus faecium, and bacterial strain CE11&#95;2 exhibited clusters of cocci-shaped cells and was identified as Pediococcus parvulus. All three candidate probiotics significantly (P < 0.05) increased transepithelial electrical resistance (TEER) in Caco-2 cells following a 3-h incubation period with hydrogen peroxide compared to control and blank groups. The combination of all three candidates as a batch culture exhibited significant efficacy in controlling intestinal colonization of ST in neonatal turkey poults. Evaluation of the combination of these selected LAB strains according to performance and intestinal health parameters of chickens and turkeys are currently in process., (© 2017 Poultry Science Association Inc.)

Three experiments were conducted to evaluate the effect of in ovo administration of FloraMax®-B11 (FM) on Marek's disease (MD) herpesvirus of turkeys (HVT) vaccine protective efficacy, hatchability, microbiota composition, morphometric analysis, and Salmonella enteritidis (SE) infection in chickens. Experiment 1 consisted of 3 trials. In trials 1 and 2, d 18 White Leghorn 15I5x71 embryos were randomly distributed in 4 groups: 1) HVT vaccinated in ovo and no Marek's disease virus (MDV) challenge; 2), HVT + FM vaccinated in ovo and no MDV challenge; 3) HVT vaccinated in ovo and challenge with virulent MDV (vMDV; strain 583A); and 4), HVT + FM vaccinated in ovo and challenge with vMDV. Trial 3 was designed exactly the same as Experiment 1 but chicks were challenged with very virulent MDV (vvMDV; strains Md5 and 612). Birds were monitored until 8 wk of age, and tested for MD incidence. Experiment 2 consisted of 3 trials. In each trial, d 18 broiler embryos were injected in ovo with either saline or FM to measure hatchability and gastrointestinal bacterial composition. In Experiment 3, d 18 broiler embryos were injected in ovo with either saline or FM. All chickens that hatched were orally gavaged with SE at hatch and kept for 7 d to monitor post-hatch BW. No significant difference (P > 0.05) between MD percentage in birds vaccinated with HVT alone or HVT + FM were observed in Experiment 1. In Experiment 2, probiotic did not negatively affect hatchability, but did reduce lactose positive Gram-negative bacteria. Further, increase in BW was associated with higher villi surface area in the ileum in chickens that received the probiotic as well as a significant reduction in the SE incidence in Experiment 3. These results suggest that in ovo administration of FM does not negatively impact the ability of HVT to protect against MD or hatchability of chickens, but improves BW during the first 7 d of life and decreases SE recovery in chickens., (© 2017 Poultry Science Association Inc.)

Fluorescein isothiocyanate dextran (FITC-d) is a 3-5 kDa marker used to measure tight junction permeability. We have previously shown that intestinal barrier function can be adversely affected by stress, poorly digested diets, or feed restriction (FR), resulting in increased intestinal inflammation-associated permeability. However, further optimization adjustments of the current FITC-d methodology are possible to enhance precision and efficacy of results in future. The objective of the present study was to optimize our current model to obtain a larger difference between control and treated groups, by optimizing the FITC-d measurement as a biomarker in a 24-h FR model to induce gut permeability in broiler chickens. One in vitro and four in vivo independent experiments were conducted. The results of the present study suggest that by increasing the dose of FITC-d (8.32 versus 4.16 mg/kg); shortening the collection time of blood samples (1 versus 2.5 h); using a pool of non-FITC-d serum as a blank, compared to previously used PBS; adding a standard curve to set a limit of detection and modifying the software's optimal sensitivity value, it was possible to obtain more consistent and reliable results.

The objectives of the present study were to evaluate the risks involved in the use of Enrofloxacin for Salmonella Enteritidis (SE) or Salmonella Heidelberg (SH) in commercial poultry and determine the effects of a probiotic as an antibiotic alternative. Two experiments were conducted to evaluate the risks involved in the use of Enrofloxacin for SE or SH in commercial poultry. Experiment 1 consisted of two trials. In each trial, chickens were assigned to one of three groups; control + SE challenged; Enrofloxacin 25 mg/kg + SE; and Enrofloxacin 50 mg/kg + SE. Chickens received Enrofloxacin in the drinking water from days 1 to 5 of age. On day 6, all groups received fresh water without any treatment. All chickens were orally gavaged with 10(7) cfu/chick of SE at 7 days of age and euthanized on 8 days of age. In Experiment 2, turkey poults were assigned to one of the three groups; control + SH; probiotic + SH; and Enrofloxacin 50 mg/kg + SH. Poults received probiotic or Enrofloxacin in the drinking water from days 1 to 5 of age. On day 6, poults received fresh water without any treatment. Poults were orally gavaged with 10(7) cfu/poult of SH at 7 days of age. Poults were weighed and humanely killed 24 h post-SH challenge to evaluate serum concentration of fluorescein isothiocyanate-dextran to evaluate intestinal permeability, metagenomics, and SH infection. In both trials of Experiment 1, chickens treated with Enrofloxacin were more susceptible to SE organ invasion and intestinal colonization when compared with control non-treated chickens (P < 0.05). In Experiment 2, poults treated with 50 mg/kg of Enrofloxacin showed an increase in body weight, however, this group also showed an increase in SH susceptibility, intestinal permeability, and lower proportion of Firmicutes and Bacteroidetes, but with control group had the highest proportion of Proteobacteria. By contrast, poults that received the probiotic had the highest proportion of Firmicutes and Bacteroidetes, but lowest Proteobacteria. The results of the present study suggest that prophylactic utilization of Enrofloxacin at five times the recommended dose in poultry increases the susceptibility to salmonellae infections, and confirms that probiotics may be an effective tool in salmonellae infections.