Use of probiotics as an alternative to formaldehyde fumigation in commercial broiler chicken hatch cabinets

Use of probiotics as an alternative to formaldehyde fumigation in commercial broiler chicken... Abstract Two experiments were conducted in a commercial broiler hatchery to evaluate the use of a spray probiotic formulation as an alternative method to control the bacterial bloom within a broiler hatch cabinet vs. formaldehyde fumigation. In Exp 1, 2 independent trials were conducted to compare hatchery sanitation between the current formaldehyde drip method vs. spray application of the probiotic. Hatchery sanitation was evaluated using the open-plate method at approximately 20% pip; 30% hatch; and 85% hatch for enumeration of total recovered non-selective aerobic bacteria (TAB); presumptive lactic acid bacteria (LAB); and total recovered Gram-negative bacteria (TGB). In Exp 2, 3 independent trials were conducted to evaluate the gastrointestinal (GIT) microbiota of neonatal chicks from hatch cabinets treated as in Exp 1. In Exp 1, in both trials, the application of the probiotic increased the number TAB and LAB present in the hatching environment (P < 0.05). Additionally, at 20% pip and 30% hatch, in both trials, there was no significant difference in TGB levels between the probiotic treatment and the formaldehyde treatment. In Exp. 2, chicks from probiotic treated hatch cabinets also showed a reduction of TGB in the GIT compared to the formaldehyde group (P < 0.05). In trial 3, the reduction in TGB persisted 24 h after hatch. The results of the present study suggest that spray application of a probiotic in commercial hatcheries can yield similar TGB levels when compared to formaldehyde early on in the hatch period. More importantly, it decreased the numbers of these bacteria within the GIT at hatch and 24 h after hatch. DESCRIPTION OF PROBLEM Under optimal conditions for temperature, nutrients, and humidity, some bacteria can theoretically divide every 20 minutes. Under these theoretical optimal conditions, a single bacterium could divide 72 times becoming 4.7 × 1021 organisms in 24 h [1]. While this theoretical maximum cannot occur in nature, it still means that one bacterium can go from being invisible to the naked eye to a readily visible colony of bacterial cells in less than a day. For microbes, near-optimal conditions are present in modern commercial poultry hatcheries. Hatch cabinets, in particular, are where some important groups of pathogens, such as Staphylococcus, Pseudomonas, Escherichia coli, Salmonella, and Aspergillus, are able to thrive [1]. Therefore, hatchery sanitation is recognized as an important factor in healthy poultry production [2]. Poor sanitization may ultimately lead to a high number of pathogenic organisms causing negative effects on hatchability and animal health leading to economic losses [3, 4]. In 1908, Pernot was the first investigator to demonstrate the use of formaldehyde fumigation of eggs and incubators as a means of controlling poultry diseases [5]. Formaldehyde (H2CO) is a gas at room temperature that is readily soluble in water and frequently used as a disinfectant or sanitizer; this is due to the fact that it is cheap, noncorrosive (in the gaseous form), and kills most viruses, bacteria (including their spores), and fungi [6, 7]. The biocidal efficacy of formaldehyde is due to its ability to act on proteins and nucleic acid bases of microorganisms. Formaldehyde also alkylates the nitrogen atoms of purine and pyrimidine bases in DNA and RNA [8]. Because of its widespread use, toxicity, and volatility, formaldehyde poses a significant danger to human health. It is an irritant for the eyes and the nose, and has a persistent noxious odor, making venting of its vapors difficult [9–11]. In 2011, the US National Toxicology Program described formaldehyde as a “known human carcinogen” [12]. An important factor in the effect of formaldehyde on the tracheal mucosa is the dissolution of the gas in mucous secretions producing a pH shift toward acidity. These changes in pH cause damage to the membrane structure and ciliary activity [13, 14]. The excessive mucus production and ciliostasis result in inadequate mucociliary action [15]. Transmission electron microscopy also has revealed shortening and loss of cilia in the epithelial cells, vacuolization, and swelling of the mitochondria, in both 18-day-old embryos and one-day-old chicks. Extending the fumigation period caused an increase in these effects [16]. Yet, in spite of these adverse effects that are extensively reported in humans and poultry, even today, most commercial hatcheries in the United States still use formaldehyde as a method to control the bacterial bloom within the hatch cabinets [17–19]. Without question, an effective hatchery sanitation program is essential for the successful operation of a poultry hatchery. In an effort to replace the use of formaldehyde, investigations have revealed large microbial populations in many hatch cabinets despite the application of alternative sanitation measures [20]. The air sampling technique we used has been used to quantitatively measure the degree of contamination by examining the microbiological loads within the hatch cabinet, and is used extensively in the poultry industry to monitor bacterial and fungal levels circulating in the air of the hatchery and to evaluate the efficiency of the decontamination measures [2, 18, 20, 21]. The purpose of the present study was to evaluate the use of a spray probiotic formulation as an alternative hatch cabinet bacterial control method vs. formaldehyde fumigation. MATERIALS AND METHODS Probiotic A specifically selected mix of 3 Bacillus subtilis isolates and 2 strains of Pediococcus acidilactici were combined and tested together as a probiotic culture in all experiments. Isolation and selection for the isolates is described below. Isolation and Selection of Lactic Acid Bacteria (LAB) Candidates Probiotic candidates were isolated from broiler chickens. Briefly, cecal epithelium, cecal lumen, and ileum epithelium were separated, homogenized, serial diluted in 0.9% sterile saline solution, and plated on de Man Rogosa Sharpe (MRS) agar plates (Catalog no. 288,110, Becton Dickinson and Co., Sparks, MD). Single colonies were obtained and identified with a number and evaluated for in vitro assessment of antimicrobial activity against Salmonella Enteritidis as previously described [22]. Two candidates were selected based on the zones of inhibition produced against the enteropathogens evaluated. These isolates were identified using API 50 CHL biochemical analysis (bioMerieux, Craponne, France) as Pediococcus acidilactici and lyophilized individually and mixed during the probiotic preparation. Isolation and Characterization of Bacillus spp. Previous research conducted in our laboratory focused on isolation of several Bacillus spp. from environmental and poultry sources [23–25]. Identification was completed using API 50 CHB biochemical analysis (bioMerieux, Craponne, France). The 3 strains were identified as Bacillus subtilis. The 3 B. subtilis strains selected were grown and sporulated individually and mixed during the probiotic preparation. Spore Preparation In an effort to grow high numbers of viable spores, a solid-state (SS) fermentation media described by Zhao et al. [26] was used. The candidate Bacillus spp. were grown in a mixture of wheat bran and rice hulls as described by Wolfenden et al. [23]. Separate Hatcher Hallway Setup All experiments were conducted in a local commercial hatchery with a capacity of 13,000 eggs per hatch cabinet. The commercial hatchery used for these experiments has 48 hatch cabinets divided into 4 separate hallways consisting of 12 hatch cabinets in each hallway. The hatchery hatches 24 hatch cabinets per day, 4 d a week. The authors did not have access to hatchability parameters for any of these studies. To prevent cross contamination by formaldehyde into the probiotic hatch cabinets, or vice versa, the treatment groups were divided into separate hallways in the hatchery. Each hallway was shut off from direct contact with the other hallways. Air was mechanically exhausted out of each hallway through the side of the building, and the air intake for each hallway was located on top of the building and was distant from the exhaust. Using separate hallways for the separate treatments prevented cross contamination between the 2 treatment groups. The hatch cabinet environment was sampled at transfer, and no bacterial growth in the hatching environment was recoverable (data not shown). Spray Application of the Probiotic The probiotic was applied 4 times through the top of the hatch cabinet using a custom-built mechanical applicator. The probiotic was applied once at transfer (19 d of incubation), and then every 10 h following the initial application until 4 total applications had occurred. Each spray application of the probiotic consisted of 30 g of the lyophilized bacteria being sprayed into the hatch cabinet. LAB were administered at approximately 108 total cfu per application. B. subtilis spores were administered at approximately 3 × 1011 spores per application. Formaldehyde-treated hatch cabinets were treated using the hatchery's standard method of formaldehyde application, which consists of drip application of 60 mL of formalin, 37% formaldehyde solution, every 3 h post transfer from the setter to the hatch cabinet. Formaldehyde treatment stopped 12 h prior to chicks being removed from the hatch cabinet. Hatch cabinet sampling time points were scheduled to be one or 2 h prior to the next application of probiotic. Probiotic-treated hatch cabinets were sampled prior to the formaldehyde-treated hatch cabinets. This was to guarantee that all of the probiotic hatch cabinets were sampled prior to the next application of probiotic. All formaldehyde- and probiotic-treated hatch cabinets were sampled within one hour. Experimental Design Experiment 1. Hatchery Sanitation Evaluation Two independent trials were conducted to compare the hatchery sanitation between the current disinfection method with formaldehyde vs. spray application of the probiotic. All hatch cabinets sampled contained embryos from the same source flock for the probiotic- and the formaldehyde-treated hatch cabinets. The only difference was the treatment that the hatch cabinet received. Hatchery sanitation was evaluated using the previously described open-plate method [21]. Each hatch cabinet had 6 carts with 15 trays of embryos per cart, with a lid covering the top tray of each cart. Each hatch cabinet had 2 fans against the front wall of the hatch cabinet between the third and fourth carts, pointed at the back of the hatch cabinet. In all experiments, 4 sampling plates of each selective media were placed into the hatch cabinets on top of the lids of the first, third, fourth, and sixth carts. Previous unpublished results had shown no difference in uniformity of plate growth if the plates were placed in the trays, below the trays, or on top of the lids of the trays. Bacteriological evaluation was conducted at approximately 20% pip, 30% hatch, and 85% hatch for enumeration of total non-selective aerobic bacteria (TAB) on Tryptic soy agar plates (TSA) (catalog no. 212,081, Becton Dickinson, Sparks, MD); LAB on MRS agar (Difco™ Lactobacilli MRS Agar VWR Cat. No. 90,004–084 Suwanee, GA); and total recovered Gram-negative bacteria (TGB) on MacConkey agar. Petri dishes containing each type of media were placed uncovered in the hatching environment for 5 minutes. Post sampling, all plates were incubated for 18 h at 37°C before the plates were enumerated. LAB and TGB enumeration was expressed in Log10 cfu based on colony counts. Incidence of total pasteurized non-selective aerobic bacteria was performed as described below. Percentage Score of Plate Coverage on TSA After TSA sampling, plates were incubated at 37°C for 18 h, and plates were scored on a percentage of plate coverage. Each individual hatch cabinet had 4 plates in it that were scored. After scoring all of the TSA sampling plates for all hatch cabinets (6 hatch cabinets per treatment group), the percentages were grouped together by treatment. To remain consistent across all sampling time points and experiments, reference pictures were used to determine the percentage of plate coverage. The reference pictures shown in Figure 1 illustrate what was scored as 0, 20, 40, 60, 80, and 100% plate coverage. If a plate did not fall directly into a category shown by the reference pictures, it was scored as a percentage somewhere between the 2 closest percentages. For instance, if a plate had more than 20% plate coverage but less than 40% plate coverage, it would be scored accordingly between 20 and 40%. Figure 1. View largeDownload slide Reference pictures used to determine the percentage of plate coverage on TSA plates placed into the hatch cabinets at all sampling time points. A) 0% plates coverage, B) 20% plate coverage, C) 40% plate coverage, D) 60% plate coverage, E) 80% plate coverage, and F) 100% plate coverage. Figure 1. View largeDownload slide Reference pictures used to determine the percentage of plate coverage on TSA plates placed into the hatch cabinets at all sampling time points. A) 0% plates coverage, B) 20% plate coverage, C) 40% plate coverage, D) 60% plate coverage, E) 80% plate coverage, and F) 100% plate coverage. Experiment 2. Evaluation of Intestinal Microbiota of Neonatal Broilers In Exp. 2, 3 independent trials were conducted to evaluate the intestinal microbiota of neonatal chicks from hatch cabinets treated with formaldehyde (current method) vs. spray application of the probiotic (n = 12/treatment). In addition, in trial 3, a sub-group of chicks (n = 12/treatment) were held for 24 h to further evaluate intestinal microbiota. As in Exp. 1, all chicks sampled in Exp. 2 came from the same source flock for both the probiotic and formaldehyde-treated hatch cabinets. Whole duodenum, ileum, and ceca were aseptically removed, separated into sterile bags, and homogenized. Samples were weighed, and 1:4 wt/vol dilutions were made with sterile 0.9% saline. Then, 10-fold dilutions of each sample from each group were made in a sterile 96-well Bacti flat-bottom plate, and the diluted samples were plated on 3 different culture media. TSA plates were utilized for enumeration of TAB in trial 1 and for enumeration of spore-forming non-selective aerobic bacteria in trials 2 and 3 (samples pasteurized by incubating sample tubes in a water bath at 70°C for 10 min [25]); MRS agar plates for total LAB; and MacConkey agar for TGB. All plates were incubated for 18 h at 37°C before bacterial count. Bacteria enumeration was expressed as Log10 cfu. All animal handling procedures were in compliance with Institutional Animal Care and Use Committee at the University of Arkansas. Statistical Analysis In all experiments, data were subjected to one-way ANOVA as a completely randomized design using the GLM procedure of SAS [27]. Data are expressed as mean ± SE, and a P-value of P < 0.05 was set as the standard for significance. RESULTS The results of the bacteriological counts recovered from hatching cabinets treated with formaldehyde or following 4applications of probiotic are summarized in Table 1 for Exp. 1. In both trials, the percentage of coverage for TAB in the probiotic-treated hatch cabinets was significantly (P < 0.05) greater than the percentage of coverage for the formaldehyde-treated hatch cabinets at all 3 sampling times, approximately 20% pip; 30% hatch, and 85% hatch. Similar results were observed in the total number of Log10 cfu of LAB. No significant differences were observed in TGB at 20% pip or 30% hatch. However, at 85% hatch, the levels of TGB in the probiotic group were significantly greater than those in the formaldehyde-treated hatch cabinets (Table 1). Table 1. Bacteriological counts recovered from hatching cabinets that received formaldehyde treatment or following 4 spray applications of probiotic – Experiment 1. 20% pip 30% hatch 85% hatch TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu Trial 1 Formaldehyde 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00 19.58 ± 1.12b 1.72 ± 0.05b 0.54 ± 0.08 37.08 ± 3.73b 2.05 ± 0.07b 0.77 ± 0.08b Probiotic 51.25 ± 4.14a 0.94 ± 0.08a 0.00 ± 0.0 100.00 ± 0.0a 2.74 ± 0.03a 0.52 ± 0.08 100.00 ± 0.0a 2.75 ± 0.03* 1.31 ± 0.06a Trial 2 Formaldehyde 0.41 ± 0.28b 0.27 ± 0.07b 0.03 ± 0.02 21.25 ± 2.72b 2.03 ± 0.05b 1.10 ± 0.06 39.58 ± 4.44b 2.16 ± 0.06b 1.35 ± 0.06b Probiotic 62.08 ± 5.03a 1.72 ± 0.06a 0.01 ± 0.01 92.08 ± 0.84a 2.46 ± 0.02a 1.22 ± 0.06 94.58 ± 1.03a 2.38 ± 0.05a 1.75 ± 0.02a 20% pip 30% hatch 85% hatch TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu Trial 1 Formaldehyde 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00 19.58 ± 1.12b 1.72 ± 0.05b 0.54 ± 0.08 37.08 ± 3.73b 2.05 ± 0.07b 0.77 ± 0.08b Probiotic 51.25 ± 4.14a 0.94 ± 0.08a 0.00 ± 0.0 100.00 ± 0.0a 2.74 ± 0.03a 0.52 ± 0.08 100.00 ± 0.0a 2.75 ± 0.03* 1.31 ± 0.06a Trial 2 Formaldehyde 0.41 ± 0.28b 0.27 ± 0.07b 0.03 ± 0.02 21.25 ± 2.72b 2.03 ± 0.05b 1.10 ± 0.06 39.58 ± 4.44b 2.16 ± 0.06b 1.35 ± 0.06b Probiotic 62.08 ± 5.03a 1.72 ± 0.06a 0.01 ± 0.01 92.08 ± 0.84a 2.46 ± 0.02a 1.22 ± 0.06 94.58 ± 1.03a 2.38 ± 0.05a 1.75 ± 0.02a a,bSuperscripts within columns in each trial between formaldehyde and probiotic treatments indicate significant difference at P < 0.05. n = 12/group. Data are expressed as mean ± SE. 1TAB: Total non-selective aerobic bacteria recovered. Data represent percentage of agar plate coverage. 2LAB: Total lactic acid bacteria recovered. Data represent Log10 cfu/plate recovered. 3TGB: Total Gram-negative bacteria recovered. Data represent Log10 cfu/plate recovered. View Large Table 1. Bacteriological counts recovered from hatching cabinets that received formaldehyde treatment or following 4 spray applications of probiotic – Experiment 1. 20% pip 30% hatch 85% hatch TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu Trial 1 Formaldehyde 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00 19.58 ± 1.12b 1.72 ± 0.05b 0.54 ± 0.08 37.08 ± 3.73b 2.05 ± 0.07b 0.77 ± 0.08b Probiotic 51.25 ± 4.14a 0.94 ± 0.08a 0.00 ± 0.0 100.00 ± 0.0a 2.74 ± 0.03a 0.52 ± 0.08 100.00 ± 0.0a 2.75 ± 0.03* 1.31 ± 0.06a Trial 2 Formaldehyde 0.41 ± 0.28b 0.27 ± 0.07b 0.03 ± 0.02 21.25 ± 2.72b 2.03 ± 0.05b 1.10 ± 0.06 39.58 ± 4.44b 2.16 ± 0.06b 1.35 ± 0.06b Probiotic 62.08 ± 5.03a 1.72 ± 0.06a 0.01 ± 0.01 92.08 ± 0.84a 2.46 ± 0.02a 1.22 ± 0.06 94.58 ± 1.03a 2.38 ± 0.05a 1.75 ± 0.02a 20% pip 30% hatch 85% hatch TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu Trial 1 Formaldehyde 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00 19.58 ± 1.12b 1.72 ± 0.05b 0.54 ± 0.08 37.08 ± 3.73b 2.05 ± 0.07b 0.77 ± 0.08b Probiotic 51.25 ± 4.14a 0.94 ± 0.08a 0.00 ± 0.0 100.00 ± 0.0a 2.74 ± 0.03a 0.52 ± 0.08 100.00 ± 0.0a 2.75 ± 0.03* 1.31 ± 0.06a Trial 2 Formaldehyde 0.41 ± 0.28b 0.27 ± 0.07b 0.03 ± 0.02 21.25 ± 2.72b 2.03 ± 0.05b 1.10 ± 0.06 39.58 ± 4.44b 2.16 ± 0.06b 1.35 ± 0.06b Probiotic 62.08 ± 5.03a 1.72 ± 0.06a 0.01 ± 0.01 92.08 ± 0.84a 2.46 ± 0.02a 1.22 ± 0.06 94.58 ± 1.03a 2.38 ± 0.05a 1.75 ± 0.02a a,bSuperscripts within columns in each trial between formaldehyde and probiotic treatments indicate significant difference at P < 0.05. n = 12/group. Data are expressed as mean ± SE. 1TAB: Total non-selective aerobic bacteria recovered. Data represent percentage of agar plate coverage. 2LAB: Total lactic acid bacteria recovered. Data represent Log10 cfu/plate recovered. 3TGB: Total Gram-negative bacteria recovered. Data represent Log10 cfu/plate recovered. View Large Table 2 summarizes the results of Log10 cfu recovered from intestinal samples on d of hatch following formaldehyde treatment or 4 applications of probiotic for all 3 trials with a 24-hour holding period for trial 3 of Exp. 2. No significant differences in the total number of Log10 cfu for the non-pasteurized TAB were observed in trial 1. However, in trial 2 and trial 3, chicks from probiotic-treated hatch cabinets showed a significant difference in pasteurized TAB when compared with formaldehyde treated chicks. For total intestinal LAB, no significant differences were observed in trials 1 and 3. Interestingly, in trial 2, a significant reduction in the total number of LAB was observed in the probiotic group when compared with the formaldehyde group. Interestingly, we observed a significant reduction in TGB in the GIT in all 3 trials in chicks from hatch cabinets treated with the probiotic, when compared with chicks from hatch cabinets that were treated with formaldehyde (Table 2). This significant reduction in TGB persisted 24 h post hatch in trial 3. Even though no significant differences were observed in LAB between treatments in the GIT, chicks from hatch cabinets treated with the probiotic and held for 24 h showed a significant increase in the total number of pasteurized TAB when compared with chicks from formaldehyde-treated hatch cabinets (Table 2). Table 2. Log10 cfu/plate recovered from intestinal samples on d of hatch following formaldehyde treatment or 4 spray applications of probiotic for all 3 trials with a 24-hour holding period for trial 3 – Experiment 2. TAB1 LAB2 TGB3 Trial 1. Day of hatch Formaldehyde 8.95 ± 0.20 7.79 ± 0.17 8.39 ± 0.19a Probiotic 8.22 ± 0.40 6.77 ± 0.56 5.79 ± 0.91b Trial 2. Day of hatch Formaldehyde 1.34 ± 0.48b 7.26 ± 0.34a 6.56 ± 0.83a Probiotic 5.30 ± 0.15a 2.88 ± 0.79b 2.60 ± 0.83b Trial 3. Day of hatch Formaldehyde 0.69 ± 0.36b 6.26 ± 0.83 3.90 ± 0.93a Probiotic 5.83 ± 0.18a 4.64 ± 0.78 1.32 ± 0.70b Trial 3. 24 h post hatch Formaldehyde 0.22 ± 0.22b 8.59 ± 0.13 8.16 ± 0.43a Probiotic 5.03 ± 0.17a 8.35 ± 0.22 5.81 ± 1.10b TAB1 LAB2 TGB3 Trial 1. Day of hatch Formaldehyde 8.95 ± 0.20 7.79 ± 0.17 8.39 ± 0.19a Probiotic 8.22 ± 0.40 6.77 ± 0.56 5.79 ± 0.91b Trial 2. Day of hatch Formaldehyde 1.34 ± 0.48b 7.26 ± 0.34a 6.56 ± 0.83a Probiotic 5.30 ± 0.15a 2.88 ± 0.79b 2.60 ± 0.83b Trial 3. Day of hatch Formaldehyde 0.69 ± 0.36b 6.26 ± 0.83 3.90 ± 0.93a Probiotic 5.83 ± 0.18a 4.64 ± 0.78 1.32 ± 0.70b Trial 3. 24 h post hatch Formaldehyde 0.22 ± 0.22b 8.59 ± 0.13 8.16 ± 0.43a Probiotic 5.03 ± 0.17a 8.35 ± 0.22 5.81 ± 1.10b a,bSuperscripts within columns in each trial between formaldehyde and probiotic treatments indicate significant difference at P < 0.05. n = 12/group. Data are expressed as mean ± SE. 1TAB: Pasteurized (Trials 2 and 3) non-selective aerobic bacteria recovered (Trial 1 was not pasteurized). 2LAB: Total lactic acid bacteria recovered. 3TGB: Total Gram-negative bacteria recovered. View Large Table 2. Log10 cfu/plate recovered from intestinal samples on d of hatch following formaldehyde treatment or 4 spray applications of probiotic for all 3 trials with a 24-hour holding period for trial 3 – Experiment 2. TAB1 LAB2 TGB3 Trial 1. Day of hatch Formaldehyde 8.95 ± 0.20 7.79 ± 0.17 8.39 ± 0.19a Probiotic 8.22 ± 0.40 6.77 ± 0.56 5.79 ± 0.91b Trial 2. Day of hatch Formaldehyde 1.34 ± 0.48b 7.26 ± 0.34a 6.56 ± 0.83a Probiotic 5.30 ± 0.15a 2.88 ± 0.79b 2.60 ± 0.83b Trial 3. Day of hatch Formaldehyde 0.69 ± 0.36b 6.26 ± 0.83 3.90 ± 0.93a Probiotic 5.83 ± 0.18a 4.64 ± 0.78 1.32 ± 0.70b Trial 3. 24 h post hatch Formaldehyde 0.22 ± 0.22b 8.59 ± 0.13 8.16 ± 0.43a Probiotic 5.03 ± 0.17a 8.35 ± 0.22 5.81 ± 1.10b TAB1 LAB2 TGB3 Trial 1. Day of hatch Formaldehyde 8.95 ± 0.20 7.79 ± 0.17 8.39 ± 0.19a Probiotic 8.22 ± 0.40 6.77 ± 0.56 5.79 ± 0.91b Trial 2. Day of hatch Formaldehyde 1.34 ± 0.48b 7.26 ± 0.34a 6.56 ± 0.83a Probiotic 5.30 ± 0.15a 2.88 ± 0.79b 2.60 ± 0.83b Trial 3. Day of hatch Formaldehyde 0.69 ± 0.36b 6.26 ± 0.83 3.90 ± 0.93a Probiotic 5.83 ± 0.18a 4.64 ± 0.78 1.32 ± 0.70b Trial 3. 24 h post hatch Formaldehyde 0.22 ± 0.22b 8.59 ± 0.13 8.16 ± 0.43a Probiotic 5.03 ± 0.17a 8.35 ± 0.22 5.81 ± 1.10b a,bSuperscripts within columns in each trial between formaldehyde and probiotic treatments indicate significant difference at P < 0.05. n = 12/group. Data are expressed as mean ± SE. 1TAB: Pasteurized (Trials 2 and 3) non-selective aerobic bacteria recovered (Trial 1 was not pasteurized). 2LAB: Total lactic acid bacteria recovered. 3TGB: Total Gram-negative bacteria recovered. View Large DISCUSSION Microbial contamination of hatching eggs is a major concern for poultry producers, as it causes poor hatchability and chick performance; hence, high standards of sanitation must be practiced in hatcheries [1]. Methods used include the application of disinfectants by wiping, spraying, and dipping, but, arguably, the most effective way of reducing the bacterial load on hatching eggs is fumigation with formaldehyde [10, 17, 28–30]. Formaldehyde is still extensively used in commercial hatcheries during the hatching period (during or just after the transfer to the hatcher). Formaldehyde, besides being an excellent anti-microbial agent, is also a toxic chemical and, as such, can seriously damage the embryo [16, 31]. In Exp. 1 of the present study, in both independent trials conducted in a commercial hatchery, the application of a defined probiotic culture containing a mix of 3 Bacillus subtilis and 2 Pediococcus acidilactici significantly increased the number of environmental TAB and LAB. However, it is interesting to observe that probiotic treatment yielded lower TGB counts in the intestinal tract of the chicks on d of hatch, and this significant reduction persisted 24 h post hatch (Table 2). E. coli, Pseudomonas pp., Salmonella spp., and many other Gram-negative organisms are present in large amounts in hatch cabinets [1, 5, 21]. The relevance of the findings in the present study is that there is good experimental and epidemiological evidence that primary infection of Gram-negative organisms is by the oral-fecal route, along with an established infectious dose [32]. Nevertheless, recent published results from our laboratory comparing intratracheal vs. oral administration of Salmonella enterica serovars Enteritidis, Typhimurium, or Seftenberg have shown that neonatal chicks can be infected via the respiratory route at a very low dose (100 cells), with cecal colonization equivalent to that recovered from a higher oral (10,000 cells) challenge [33–36]. Understanding the anatomical and immunological defenses of the avian respiratory tract helps to clarify this issue. Architecture of the avian respiratory tract is an important component to susceptibility and resistance to infectious agents. In day-old chicks and turkeys, no or very few infiltrating lymphocytes are seen in the primary bronchi region [37, 38], and it is not until 3 to 4 wk of age that the lymphoid nodules are developed at these locations [39, 40]. During the following wk, the number of IgG-, IgA-, or IgM-producing cells continues to increase- however, the bronchial-associated lymphoid tissue is not mature until chickens are 6 to 8 wk old [41–43]. Hence, commercial neonatal poultry are extremely susceptible to airborne pathogens, regardless of whether or not they are respiratory or enteric bacteria [44, 45]. On the other hand, these chicks are deprived of acquiring their natural microbiota that colonize their GIT immediately after hatching under natural conditions. Today, the microbiome is recognized as the “forgotten organ,” operating like an organ within the host and orchestrating numerous physiological and biological functions that have a profound impact on the balance between health and disease [46, 47]. Early establishment of the microbiome has been reported to improve the assembly of the gut-associated lymphoid tissue [48], mediate in the development of the immune system [49], maintain mucosal barrier integrity [50], modulate proliferation of enterocytes [51], adjust blood flow [52], regulate the enteric nervous system [53], and improve digestion of nutrients [54–56]. Essential colonization of these bacterial populations starts at birth/hatch, and is followed by progressive assembly of a complex and dynamic microbial community [57]. 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Google Scholar CrossRef Search ADS PubMed Acknowledgments This research was supported by the Arkansas Bioscience Institute under the project of Development of an Avian Model for Evaluation Early Enteric Microbial Colonization on the Gastrointestinal Tract and Immune Function. © 2018 Poultry Science Association Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Poultry Research Oxford University Press

Use of probiotics as an alternative to formaldehyde fumigation in commercial broiler chicken hatch cabinets

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Applied Poultry Science, Inc.
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© 2018 Poultry Science Association Inc.
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1056-6171
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1537-0437
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10.3382/japr/pfy008
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Abstract

Abstract Two experiments were conducted in a commercial broiler hatchery to evaluate the use of a spray probiotic formulation as an alternative method to control the bacterial bloom within a broiler hatch cabinet vs. formaldehyde fumigation. In Exp 1, 2 independent trials were conducted to compare hatchery sanitation between the current formaldehyde drip method vs. spray application of the probiotic. Hatchery sanitation was evaluated using the open-plate method at approximately 20% pip; 30% hatch; and 85% hatch for enumeration of total recovered non-selective aerobic bacteria (TAB); presumptive lactic acid bacteria (LAB); and total recovered Gram-negative bacteria (TGB). In Exp 2, 3 independent trials were conducted to evaluate the gastrointestinal (GIT) microbiota of neonatal chicks from hatch cabinets treated as in Exp 1. In Exp 1, in both trials, the application of the probiotic increased the number TAB and LAB present in the hatching environment (P < 0.05). Additionally, at 20% pip and 30% hatch, in both trials, there was no significant difference in TGB levels between the probiotic treatment and the formaldehyde treatment. In Exp. 2, chicks from probiotic treated hatch cabinets also showed a reduction of TGB in the GIT compared to the formaldehyde group (P < 0.05). In trial 3, the reduction in TGB persisted 24 h after hatch. The results of the present study suggest that spray application of a probiotic in commercial hatcheries can yield similar TGB levels when compared to formaldehyde early on in the hatch period. More importantly, it decreased the numbers of these bacteria within the GIT at hatch and 24 h after hatch. DESCRIPTION OF PROBLEM Under optimal conditions for temperature, nutrients, and humidity, some bacteria can theoretically divide every 20 minutes. Under these theoretical optimal conditions, a single bacterium could divide 72 times becoming 4.7 × 1021 organisms in 24 h [1]. While this theoretical maximum cannot occur in nature, it still means that one bacterium can go from being invisible to the naked eye to a readily visible colony of bacterial cells in less than a day. For microbes, near-optimal conditions are present in modern commercial poultry hatcheries. Hatch cabinets, in particular, are where some important groups of pathogens, such as Staphylococcus, Pseudomonas, Escherichia coli, Salmonella, and Aspergillus, are able to thrive [1]. Therefore, hatchery sanitation is recognized as an important factor in healthy poultry production [2]. Poor sanitization may ultimately lead to a high number of pathogenic organisms causing negative effects on hatchability and animal health leading to economic losses [3, 4]. In 1908, Pernot was the first investigator to demonstrate the use of formaldehyde fumigation of eggs and incubators as a means of controlling poultry diseases [5]. Formaldehyde (H2CO) is a gas at room temperature that is readily soluble in water and frequently used as a disinfectant or sanitizer; this is due to the fact that it is cheap, noncorrosive (in the gaseous form), and kills most viruses, bacteria (including their spores), and fungi [6, 7]. The biocidal efficacy of formaldehyde is due to its ability to act on proteins and nucleic acid bases of microorganisms. Formaldehyde also alkylates the nitrogen atoms of purine and pyrimidine bases in DNA and RNA [8]. Because of its widespread use, toxicity, and volatility, formaldehyde poses a significant danger to human health. It is an irritant for the eyes and the nose, and has a persistent noxious odor, making venting of its vapors difficult [9–11]. In 2011, the US National Toxicology Program described formaldehyde as a “known human carcinogen” [12]. An important factor in the effect of formaldehyde on the tracheal mucosa is the dissolution of the gas in mucous secretions producing a pH shift toward acidity. These changes in pH cause damage to the membrane structure and ciliary activity [13, 14]. The excessive mucus production and ciliostasis result in inadequate mucociliary action [15]. Transmission electron microscopy also has revealed shortening and loss of cilia in the epithelial cells, vacuolization, and swelling of the mitochondria, in both 18-day-old embryos and one-day-old chicks. Extending the fumigation period caused an increase in these effects [16]. Yet, in spite of these adverse effects that are extensively reported in humans and poultry, even today, most commercial hatcheries in the United States still use formaldehyde as a method to control the bacterial bloom within the hatch cabinets [17–19]. Without question, an effective hatchery sanitation program is essential for the successful operation of a poultry hatchery. In an effort to replace the use of formaldehyde, investigations have revealed large microbial populations in many hatch cabinets despite the application of alternative sanitation measures [20]. The air sampling technique we used has been used to quantitatively measure the degree of contamination by examining the microbiological loads within the hatch cabinet, and is used extensively in the poultry industry to monitor bacterial and fungal levels circulating in the air of the hatchery and to evaluate the efficiency of the decontamination measures [2, 18, 20, 21]. The purpose of the present study was to evaluate the use of a spray probiotic formulation as an alternative hatch cabinet bacterial control method vs. formaldehyde fumigation. MATERIALS AND METHODS Probiotic A specifically selected mix of 3 Bacillus subtilis isolates and 2 strains of Pediococcus acidilactici were combined and tested together as a probiotic culture in all experiments. Isolation and selection for the isolates is described below. Isolation and Selection of Lactic Acid Bacteria (LAB) Candidates Probiotic candidates were isolated from broiler chickens. Briefly, cecal epithelium, cecal lumen, and ileum epithelium were separated, homogenized, serial diluted in 0.9% sterile saline solution, and plated on de Man Rogosa Sharpe (MRS) agar plates (Catalog no. 288,110, Becton Dickinson and Co., Sparks, MD). Single colonies were obtained and identified with a number and evaluated for in vitro assessment of antimicrobial activity against Salmonella Enteritidis as previously described [22]. Two candidates were selected based on the zones of inhibition produced against the enteropathogens evaluated. These isolates were identified using API 50 CHL biochemical analysis (bioMerieux, Craponne, France) as Pediococcus acidilactici and lyophilized individually and mixed during the probiotic preparation. Isolation and Characterization of Bacillus spp. Previous research conducted in our laboratory focused on isolation of several Bacillus spp. from environmental and poultry sources [23–25]. Identification was completed using API 50 CHB biochemical analysis (bioMerieux, Craponne, France). The 3 strains were identified as Bacillus subtilis. The 3 B. subtilis strains selected were grown and sporulated individually and mixed during the probiotic preparation. Spore Preparation In an effort to grow high numbers of viable spores, a solid-state (SS) fermentation media described by Zhao et al. [26] was used. The candidate Bacillus spp. were grown in a mixture of wheat bran and rice hulls as described by Wolfenden et al. [23]. Separate Hatcher Hallway Setup All experiments were conducted in a local commercial hatchery with a capacity of 13,000 eggs per hatch cabinet. The commercial hatchery used for these experiments has 48 hatch cabinets divided into 4 separate hallways consisting of 12 hatch cabinets in each hallway. The hatchery hatches 24 hatch cabinets per day, 4 d a week. The authors did not have access to hatchability parameters for any of these studies. To prevent cross contamination by formaldehyde into the probiotic hatch cabinets, or vice versa, the treatment groups were divided into separate hallways in the hatchery. Each hallway was shut off from direct contact with the other hallways. Air was mechanically exhausted out of each hallway through the side of the building, and the air intake for each hallway was located on top of the building and was distant from the exhaust. Using separate hallways for the separate treatments prevented cross contamination between the 2 treatment groups. The hatch cabinet environment was sampled at transfer, and no bacterial growth in the hatching environment was recoverable (data not shown). Spray Application of the Probiotic The probiotic was applied 4 times through the top of the hatch cabinet using a custom-built mechanical applicator. The probiotic was applied once at transfer (19 d of incubation), and then every 10 h following the initial application until 4 total applications had occurred. Each spray application of the probiotic consisted of 30 g of the lyophilized bacteria being sprayed into the hatch cabinet. LAB were administered at approximately 108 total cfu per application. B. subtilis spores were administered at approximately 3 × 1011 spores per application. Formaldehyde-treated hatch cabinets were treated using the hatchery's standard method of formaldehyde application, which consists of drip application of 60 mL of formalin, 37% formaldehyde solution, every 3 h post transfer from the setter to the hatch cabinet. Formaldehyde treatment stopped 12 h prior to chicks being removed from the hatch cabinet. Hatch cabinet sampling time points were scheduled to be one or 2 h prior to the next application of probiotic. Probiotic-treated hatch cabinets were sampled prior to the formaldehyde-treated hatch cabinets. This was to guarantee that all of the probiotic hatch cabinets were sampled prior to the next application of probiotic. All formaldehyde- and probiotic-treated hatch cabinets were sampled within one hour. Experimental Design Experiment 1. Hatchery Sanitation Evaluation Two independent trials were conducted to compare the hatchery sanitation between the current disinfection method with formaldehyde vs. spray application of the probiotic. All hatch cabinets sampled contained embryos from the same source flock for the probiotic- and the formaldehyde-treated hatch cabinets. The only difference was the treatment that the hatch cabinet received. Hatchery sanitation was evaluated using the previously described open-plate method [21]. Each hatch cabinet had 6 carts with 15 trays of embryos per cart, with a lid covering the top tray of each cart. Each hatch cabinet had 2 fans against the front wall of the hatch cabinet between the third and fourth carts, pointed at the back of the hatch cabinet. In all experiments, 4 sampling plates of each selective media were placed into the hatch cabinets on top of the lids of the first, third, fourth, and sixth carts. Previous unpublished results had shown no difference in uniformity of plate growth if the plates were placed in the trays, below the trays, or on top of the lids of the trays. Bacteriological evaluation was conducted at approximately 20% pip, 30% hatch, and 85% hatch for enumeration of total non-selective aerobic bacteria (TAB) on Tryptic soy agar plates (TSA) (catalog no. 212,081, Becton Dickinson, Sparks, MD); LAB on MRS agar (Difco™ Lactobacilli MRS Agar VWR Cat. No. 90,004–084 Suwanee, GA); and total recovered Gram-negative bacteria (TGB) on MacConkey agar. Petri dishes containing each type of media were placed uncovered in the hatching environment for 5 minutes. Post sampling, all plates were incubated for 18 h at 37°C before the plates were enumerated. LAB and TGB enumeration was expressed in Log10 cfu based on colony counts. Incidence of total pasteurized non-selective aerobic bacteria was performed as described below. Percentage Score of Plate Coverage on TSA After TSA sampling, plates were incubated at 37°C for 18 h, and plates were scored on a percentage of plate coverage. Each individual hatch cabinet had 4 plates in it that were scored. After scoring all of the TSA sampling plates for all hatch cabinets (6 hatch cabinets per treatment group), the percentages were grouped together by treatment. To remain consistent across all sampling time points and experiments, reference pictures were used to determine the percentage of plate coverage. The reference pictures shown in Figure 1 illustrate what was scored as 0, 20, 40, 60, 80, and 100% plate coverage. If a plate did not fall directly into a category shown by the reference pictures, it was scored as a percentage somewhere between the 2 closest percentages. For instance, if a plate had more than 20% plate coverage but less than 40% plate coverage, it would be scored accordingly between 20 and 40%. Figure 1. View largeDownload slide Reference pictures used to determine the percentage of plate coverage on TSA plates placed into the hatch cabinets at all sampling time points. A) 0% plates coverage, B) 20% plate coverage, C) 40% plate coverage, D) 60% plate coverage, E) 80% plate coverage, and F) 100% plate coverage. Figure 1. View largeDownload slide Reference pictures used to determine the percentage of plate coverage on TSA plates placed into the hatch cabinets at all sampling time points. A) 0% plates coverage, B) 20% plate coverage, C) 40% plate coverage, D) 60% plate coverage, E) 80% plate coverage, and F) 100% plate coverage. Experiment 2. Evaluation of Intestinal Microbiota of Neonatal Broilers In Exp. 2, 3 independent trials were conducted to evaluate the intestinal microbiota of neonatal chicks from hatch cabinets treated with formaldehyde (current method) vs. spray application of the probiotic (n = 12/treatment). In addition, in trial 3, a sub-group of chicks (n = 12/treatment) were held for 24 h to further evaluate intestinal microbiota. As in Exp. 1, all chicks sampled in Exp. 2 came from the same source flock for both the probiotic and formaldehyde-treated hatch cabinets. Whole duodenum, ileum, and ceca were aseptically removed, separated into sterile bags, and homogenized. Samples were weighed, and 1:4 wt/vol dilutions were made with sterile 0.9% saline. Then, 10-fold dilutions of each sample from each group were made in a sterile 96-well Bacti flat-bottom plate, and the diluted samples were plated on 3 different culture media. TSA plates were utilized for enumeration of TAB in trial 1 and for enumeration of spore-forming non-selective aerobic bacteria in trials 2 and 3 (samples pasteurized by incubating sample tubes in a water bath at 70°C for 10 min [25]); MRS agar plates for total LAB; and MacConkey agar for TGB. All plates were incubated for 18 h at 37°C before bacterial count. Bacteria enumeration was expressed as Log10 cfu. All animal handling procedures were in compliance with Institutional Animal Care and Use Committee at the University of Arkansas. Statistical Analysis In all experiments, data were subjected to one-way ANOVA as a completely randomized design using the GLM procedure of SAS [27]. Data are expressed as mean ± SE, and a P-value of P < 0.05 was set as the standard for significance. RESULTS The results of the bacteriological counts recovered from hatching cabinets treated with formaldehyde or following 4applications of probiotic are summarized in Table 1 for Exp. 1. In both trials, the percentage of coverage for TAB in the probiotic-treated hatch cabinets was significantly (P < 0.05) greater than the percentage of coverage for the formaldehyde-treated hatch cabinets at all 3 sampling times, approximately 20% pip; 30% hatch, and 85% hatch. Similar results were observed in the total number of Log10 cfu of LAB. No significant differences were observed in TGB at 20% pip or 30% hatch. However, at 85% hatch, the levels of TGB in the probiotic group were significantly greater than those in the formaldehyde-treated hatch cabinets (Table 1). Table 1. Bacteriological counts recovered from hatching cabinets that received formaldehyde treatment or following 4 spray applications of probiotic – Experiment 1. 20% pip 30% hatch 85% hatch TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu Trial 1 Formaldehyde 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00 19.58 ± 1.12b 1.72 ± 0.05b 0.54 ± 0.08 37.08 ± 3.73b 2.05 ± 0.07b 0.77 ± 0.08b Probiotic 51.25 ± 4.14a 0.94 ± 0.08a 0.00 ± 0.0 100.00 ± 0.0a 2.74 ± 0.03a 0.52 ± 0.08 100.00 ± 0.0a 2.75 ± 0.03* 1.31 ± 0.06a Trial 2 Formaldehyde 0.41 ± 0.28b 0.27 ± 0.07b 0.03 ± 0.02 21.25 ± 2.72b 2.03 ± 0.05b 1.10 ± 0.06 39.58 ± 4.44b 2.16 ± 0.06b 1.35 ± 0.06b Probiotic 62.08 ± 5.03a 1.72 ± 0.06a 0.01 ± 0.01 92.08 ± 0.84a 2.46 ± 0.02a 1.22 ± 0.06 94.58 ± 1.03a 2.38 ± 0.05a 1.75 ± 0.02a 20% pip 30% hatch 85% hatch TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu Trial 1 Formaldehyde 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00 19.58 ± 1.12b 1.72 ± 0.05b 0.54 ± 0.08 37.08 ± 3.73b 2.05 ± 0.07b 0.77 ± 0.08b Probiotic 51.25 ± 4.14a 0.94 ± 0.08a 0.00 ± 0.0 100.00 ± 0.0a 2.74 ± 0.03a 0.52 ± 0.08 100.00 ± 0.0a 2.75 ± 0.03* 1.31 ± 0.06a Trial 2 Formaldehyde 0.41 ± 0.28b 0.27 ± 0.07b 0.03 ± 0.02 21.25 ± 2.72b 2.03 ± 0.05b 1.10 ± 0.06 39.58 ± 4.44b 2.16 ± 0.06b 1.35 ± 0.06b Probiotic 62.08 ± 5.03a 1.72 ± 0.06a 0.01 ± 0.01 92.08 ± 0.84a 2.46 ± 0.02a 1.22 ± 0.06 94.58 ± 1.03a 2.38 ± 0.05a 1.75 ± 0.02a a,bSuperscripts within columns in each trial between formaldehyde and probiotic treatments indicate significant difference at P < 0.05. n = 12/group. Data are expressed as mean ± SE. 1TAB: Total non-selective aerobic bacteria recovered. Data represent percentage of agar plate coverage. 2LAB: Total lactic acid bacteria recovered. Data represent Log10 cfu/plate recovered. 3TGB: Total Gram-negative bacteria recovered. Data represent Log10 cfu/plate recovered. View Large Table 1. Bacteriological counts recovered from hatching cabinets that received formaldehyde treatment or following 4 spray applications of probiotic – Experiment 1. 20% pip 30% hatch 85% hatch TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu Trial 1 Formaldehyde 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00 19.58 ± 1.12b 1.72 ± 0.05b 0.54 ± 0.08 37.08 ± 3.73b 2.05 ± 0.07b 0.77 ± 0.08b Probiotic 51.25 ± 4.14a 0.94 ± 0.08a 0.00 ± 0.0 100.00 ± 0.0a 2.74 ± 0.03a 0.52 ± 0.08 100.00 ± 0.0a 2.75 ± 0.03* 1.31 ± 0.06a Trial 2 Formaldehyde 0.41 ± 0.28b 0.27 ± 0.07b 0.03 ± 0.02 21.25 ± 2.72b 2.03 ± 0.05b 1.10 ± 0.06 39.58 ± 4.44b 2.16 ± 0.06b 1.35 ± 0.06b Probiotic 62.08 ± 5.03a 1.72 ± 0.06a 0.01 ± 0.01 92.08 ± 0.84a 2.46 ± 0.02a 1.22 ± 0.06 94.58 ± 1.03a 2.38 ± 0.05a 1.75 ± 0.02a 20% pip 30% hatch 85% hatch TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu TAB1% LAB 2 Log10 cfu TGB 3 Log10 cfu Trial 1 Formaldehyde 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00 19.58 ± 1.12b 1.72 ± 0.05b 0.54 ± 0.08 37.08 ± 3.73b 2.05 ± 0.07b 0.77 ± 0.08b Probiotic 51.25 ± 4.14a 0.94 ± 0.08a 0.00 ± 0.0 100.00 ± 0.0a 2.74 ± 0.03a 0.52 ± 0.08 100.00 ± 0.0a 2.75 ± 0.03* 1.31 ± 0.06a Trial 2 Formaldehyde 0.41 ± 0.28b 0.27 ± 0.07b 0.03 ± 0.02 21.25 ± 2.72b 2.03 ± 0.05b 1.10 ± 0.06 39.58 ± 4.44b 2.16 ± 0.06b 1.35 ± 0.06b Probiotic 62.08 ± 5.03a 1.72 ± 0.06a 0.01 ± 0.01 92.08 ± 0.84a 2.46 ± 0.02a 1.22 ± 0.06 94.58 ± 1.03a 2.38 ± 0.05a 1.75 ± 0.02a a,bSuperscripts within columns in each trial between formaldehyde and probiotic treatments indicate significant difference at P < 0.05. n = 12/group. Data are expressed as mean ± SE. 1TAB: Total non-selective aerobic bacteria recovered. Data represent percentage of agar plate coverage. 2LAB: Total lactic acid bacteria recovered. Data represent Log10 cfu/plate recovered. 3TGB: Total Gram-negative bacteria recovered. Data represent Log10 cfu/plate recovered. View Large Table 2 summarizes the results of Log10 cfu recovered from intestinal samples on d of hatch following formaldehyde treatment or 4 applications of probiotic for all 3 trials with a 24-hour holding period for trial 3 of Exp. 2. No significant differences in the total number of Log10 cfu for the non-pasteurized TAB were observed in trial 1. However, in trial 2 and trial 3, chicks from probiotic-treated hatch cabinets showed a significant difference in pasteurized TAB when compared with formaldehyde treated chicks. For total intestinal LAB, no significant differences were observed in trials 1 and 3. Interestingly, in trial 2, a significant reduction in the total number of LAB was observed in the probiotic group when compared with the formaldehyde group. Interestingly, we observed a significant reduction in TGB in the GIT in all 3 trials in chicks from hatch cabinets treated with the probiotic, when compared with chicks from hatch cabinets that were treated with formaldehyde (Table 2). This significant reduction in TGB persisted 24 h post hatch in trial 3. Even though no significant differences were observed in LAB between treatments in the GIT, chicks from hatch cabinets treated with the probiotic and held for 24 h showed a significant increase in the total number of pasteurized TAB when compared with chicks from formaldehyde-treated hatch cabinets (Table 2). Table 2. Log10 cfu/plate recovered from intestinal samples on d of hatch following formaldehyde treatment or 4 spray applications of probiotic for all 3 trials with a 24-hour holding period for trial 3 – Experiment 2. TAB1 LAB2 TGB3 Trial 1. Day of hatch Formaldehyde 8.95 ± 0.20 7.79 ± 0.17 8.39 ± 0.19a Probiotic 8.22 ± 0.40 6.77 ± 0.56 5.79 ± 0.91b Trial 2. Day of hatch Formaldehyde 1.34 ± 0.48b 7.26 ± 0.34a 6.56 ± 0.83a Probiotic 5.30 ± 0.15a 2.88 ± 0.79b 2.60 ± 0.83b Trial 3. Day of hatch Formaldehyde 0.69 ± 0.36b 6.26 ± 0.83 3.90 ± 0.93a Probiotic 5.83 ± 0.18a 4.64 ± 0.78 1.32 ± 0.70b Trial 3. 24 h post hatch Formaldehyde 0.22 ± 0.22b 8.59 ± 0.13 8.16 ± 0.43a Probiotic 5.03 ± 0.17a 8.35 ± 0.22 5.81 ± 1.10b TAB1 LAB2 TGB3 Trial 1. Day of hatch Formaldehyde 8.95 ± 0.20 7.79 ± 0.17 8.39 ± 0.19a Probiotic 8.22 ± 0.40 6.77 ± 0.56 5.79 ± 0.91b Trial 2. Day of hatch Formaldehyde 1.34 ± 0.48b 7.26 ± 0.34a 6.56 ± 0.83a Probiotic 5.30 ± 0.15a 2.88 ± 0.79b 2.60 ± 0.83b Trial 3. Day of hatch Formaldehyde 0.69 ± 0.36b 6.26 ± 0.83 3.90 ± 0.93a Probiotic 5.83 ± 0.18a 4.64 ± 0.78 1.32 ± 0.70b Trial 3. 24 h post hatch Formaldehyde 0.22 ± 0.22b 8.59 ± 0.13 8.16 ± 0.43a Probiotic 5.03 ± 0.17a 8.35 ± 0.22 5.81 ± 1.10b a,bSuperscripts within columns in each trial between formaldehyde and probiotic treatments indicate significant difference at P < 0.05. n = 12/group. Data are expressed as mean ± SE. 1TAB: Pasteurized (Trials 2 and 3) non-selective aerobic bacteria recovered (Trial 1 was not pasteurized). 2LAB: Total lactic acid bacteria recovered. 3TGB: Total Gram-negative bacteria recovered. View Large Table 2. Log10 cfu/plate recovered from intestinal samples on d of hatch following formaldehyde treatment or 4 spray applications of probiotic for all 3 trials with a 24-hour holding period for trial 3 – Experiment 2. TAB1 LAB2 TGB3 Trial 1. Day of hatch Formaldehyde 8.95 ± 0.20 7.79 ± 0.17 8.39 ± 0.19a Probiotic 8.22 ± 0.40 6.77 ± 0.56 5.79 ± 0.91b Trial 2. Day of hatch Formaldehyde 1.34 ± 0.48b 7.26 ± 0.34a 6.56 ± 0.83a Probiotic 5.30 ± 0.15a 2.88 ± 0.79b 2.60 ± 0.83b Trial 3. Day of hatch Formaldehyde 0.69 ± 0.36b 6.26 ± 0.83 3.90 ± 0.93a Probiotic 5.83 ± 0.18a 4.64 ± 0.78 1.32 ± 0.70b Trial 3. 24 h post hatch Formaldehyde 0.22 ± 0.22b 8.59 ± 0.13 8.16 ± 0.43a Probiotic 5.03 ± 0.17a 8.35 ± 0.22 5.81 ± 1.10b TAB1 LAB2 TGB3 Trial 1. Day of hatch Formaldehyde 8.95 ± 0.20 7.79 ± 0.17 8.39 ± 0.19a Probiotic 8.22 ± 0.40 6.77 ± 0.56 5.79 ± 0.91b Trial 2. Day of hatch Formaldehyde 1.34 ± 0.48b 7.26 ± 0.34a 6.56 ± 0.83a Probiotic 5.30 ± 0.15a 2.88 ± 0.79b 2.60 ± 0.83b Trial 3. Day of hatch Formaldehyde 0.69 ± 0.36b 6.26 ± 0.83 3.90 ± 0.93a Probiotic 5.83 ± 0.18a 4.64 ± 0.78 1.32 ± 0.70b Trial 3. 24 h post hatch Formaldehyde 0.22 ± 0.22b 8.59 ± 0.13 8.16 ± 0.43a Probiotic 5.03 ± 0.17a 8.35 ± 0.22 5.81 ± 1.10b a,bSuperscripts within columns in each trial between formaldehyde and probiotic treatments indicate significant difference at P < 0.05. n = 12/group. Data are expressed as mean ± SE. 1TAB: Pasteurized (Trials 2 and 3) non-selective aerobic bacteria recovered (Trial 1 was not pasteurized). 2LAB: Total lactic acid bacteria recovered. 3TGB: Total Gram-negative bacteria recovered. View Large DISCUSSION Microbial contamination of hatching eggs is a major concern for poultry producers, as it causes poor hatchability and chick performance; hence, high standards of sanitation must be practiced in hatcheries [1]. Methods used include the application of disinfectants by wiping, spraying, and dipping, but, arguably, the most effective way of reducing the bacterial load on hatching eggs is fumigation with formaldehyde [10, 17, 28–30]. Formaldehyde is still extensively used in commercial hatcheries during the hatching period (during or just after the transfer to the hatcher). Formaldehyde, besides being an excellent anti-microbial agent, is also a toxic chemical and, as such, can seriously damage the embryo [16, 31]. In Exp. 1 of the present study, in both independent trials conducted in a commercial hatchery, the application of a defined probiotic culture containing a mix of 3 Bacillus subtilis and 2 Pediococcus acidilactici significantly increased the number of environmental TAB and LAB. However, it is interesting to observe that probiotic treatment yielded lower TGB counts in the intestinal tract of the chicks on d of hatch, and this significant reduction persisted 24 h post hatch (Table 2). E. coli, Pseudomonas pp., Salmonella spp., and many other Gram-negative organisms are present in large amounts in hatch cabinets [1, 5, 21]. The relevance of the findings in the present study is that there is good experimental and epidemiological evidence that primary infection of Gram-negative organisms is by the oral-fecal route, along with an established infectious dose [32]. Nevertheless, recent published results from our laboratory comparing intratracheal vs. oral administration of Salmonella enterica serovars Enteritidis, Typhimurium, or Seftenberg have shown that neonatal chicks can be infected via the respiratory route at a very low dose (100 cells), with cecal colonization equivalent to that recovered from a higher oral (10,000 cells) challenge [33–36]. Understanding the anatomical and immunological defenses of the avian respiratory tract helps to clarify this issue. Architecture of the avian respiratory tract is an important component to susceptibility and resistance to infectious agents. In day-old chicks and turkeys, no or very few infiltrating lymphocytes are seen in the primary bronchi region [37, 38], and it is not until 3 to 4 wk of age that the lymphoid nodules are developed at these locations [39, 40]. During the following wk, the number of IgG-, IgA-, or IgM-producing cells continues to increase- however, the bronchial-associated lymphoid tissue is not mature until chickens are 6 to 8 wk old [41–43]. Hence, commercial neonatal poultry are extremely susceptible to airborne pathogens, regardless of whether or not they are respiratory or enteric bacteria [44, 45]. On the other hand, these chicks are deprived of acquiring their natural microbiota that colonize their GIT immediately after hatching under natural conditions. Today, the microbiome is recognized as the “forgotten organ,” operating like an organ within the host and orchestrating numerous physiological and biological functions that have a profound impact on the balance between health and disease [46, 47]. Early establishment of the microbiome has been reported to improve the assembly of the gut-associated lymphoid tissue [48], mediate in the development of the immune system [49], maintain mucosal barrier integrity [50], modulate proliferation of enterocytes [51], adjust blood flow [52], regulate the enteric nervous system [53], and improve digestion of nutrients [54–56]. Essential colonization of these bacterial populations starts at birth/hatch, and is followed by progressive assembly of a complex and dynamic microbial community [57]. 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Journal of Applied Poultry ResearchOxford University Press

Published: Mar 16, 2018

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